Protein-coupled receptor

ABSTRACT

The present application provides a gene encoding a G protein-coupled receptor termed nGPCR-14; constructs and recombinant host cells incorporating the genes; the nGPCR-14 polypeptides encoded by the gene; antibodies to the nGPCR-x polypeptides; and methods of making and using all of the foregoing.

FIELD OF THE INVENTION

The present invention relates generally to the fields of genetics andcellular and molecular biology. More particularly, the invention relatesto novel G protein coupled receptors, to polynucleotides that encodesuch novel receptors, to reagents such as antibodies, probes, primersand kits comprising such antibodies, probes, primers related to thesame, and to methods which use the novel G protein coupled receptors,polynucleotides or reagents.

BACKGROUND OF THE INVENTION

The G protein-coupled receptors (GPCRs) form a vast superfamily of cellsurface receptors which are characterized by an amino-terminalextracellular domain, a carboxyl terminal intracellular domain, and aserpentine structure that passes through the cell membrane seven times.Hence, such receptors are sometimes also referred to as seventransmembrane (7TM) receptors. These seven transmembrane domains definethree extracellular loops and three intracellular loops, in addition tothe amino- and carboxy-terminal domains. The extracellular portions ofthe receptor have a role in recognizing and binding one or moreextracellular binding partners (e.g., ligands), whereas theintracellular portions have a role in recognizing and communicating withdownstream molecules in the signal transduction cascade.

The G protein-coupled receptors bind a variety of ligands includingcalcium ions, hormones, chemokines, neuropeptides, neurotransmitters,nucleotides, lipids, odorants, and even photons, and are important inthe normal (and sometimes the aberrant) function of many cell types.[See generally Strosberg, Eur. J. Biochem. 196:1-10 (1991) and Bohm etal., Biochem J. 322:1-18 (1997).] When a specific ligand binds to itscorresponding receptor, the ligand typically stimulates the receptor toactivate a specific heterotrimeric guanine-nucleotide-binding regulatoryprotein (G-protein) that is coupled to the intracellular portion of thereceptor. The G protein in turn transmits a signal to an effectormolecule within the cell, by either stimulating or inhibiting theactivity of that effector molecule. These effector molecules includeadenylate cyclase, phospholipases and ion channels. Adenylate cyclaseand phospholipases are enzymes that are involved in the production ofthe second messenger molecules cAMP, inositol triphosphate anddiacyglycerol. It is through this sequence of events that anextracellular ligand stimuli exerts intracellular changes through a Gprotein-coupled receptor. Each such receptor has its own characteristicprimary structure, expression pattern, ligand-binding profile, andintracellular effector system.

Because of the vital role of G protein-coupled receptors in thecommunication between cells and their environment, such receptors areattractive targets for therapeutic intervention, for example byactivating or antagonizing such receptors. For receptors having a knownligand, the identification of agonists or antagonists may be soughtspecifically to enhance or inhibit the action of the ligand. Some Gprotein-coupled receptors have roles in disease pathogenesis (e.g.,certain chemokine receptors that act as HIV co-receptors may have a rolein AIDS pathogenesis), and are attractive targets for therapeuticintervention even in the absence of knowledge of the natural ligand ofthe receptor. Other receptors are attractive targets for therapeuticintervention by virtue of their expression pattern in tissues or celltypes that are themselves attractive targets for therapeuticintervention. Examples of this latter category of receptors includereceptors expressed in immune cells, which can be targeted to eitherinhibit autoimmune responses or to enhance immune responses to fightpathogens or cancer; and receptors expressed in the brain or otherneural organs and tissues, which are likely targets in the treatment ofschizophrenia, depression, bipolar disease, or other neurologicaldisorders. This latter category of receptor is also useful as a markerfor identifying and/or purifying (e.g., via fluorescence-activated cellsorting) cellular subtypes that express the receptor. Unfortunately,only a limited number of G protein receptors from the central nervoussystem (CNS) are known. Thus, a need exists for G protein-coupledreceptors that have been identified and show promise as targets fortherapeutic intervention in a variety of animals, including humans.

SUMMARY OF THE INVENTION

The present invention relates to an isolated nucleic acid molecule thatcomprises a nucleotide sequence that encodes a polypeptide comprising anamino acid sequence homologous to even numbered sequences ranging fromSEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or afragment thereof. The nucleic acid molecule encodes at least a portionof nGPCR-x. In some embodiments, the nucleic acid molecule comprises asequence that encodes a polypeptide comprising even numbered sequencesranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ IDNO: 192, or a fragment thereof. In some embodiments, the nucleic acidmolecule comprises a sequence homologous to odd numbered sequencesranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ IDNO: 191, or a fragment thereof. In some embodiments, the nucleic acidmolecule comprises a sequence selected from the group consisting of oddnumbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ IDNO: 185 and SEQ ID NO: 191, and fragments thereof.

According to some embodiments, the present invention provides vectorswhich comprise the nucleic acid molecule of the invention. In someembodiments, the vector is an expression vector.

According to some embodiments, the present invention provides host cellswhich comprise the vectors of the invention. In some embodiments, thehost cells comprise expression vectors.

The present invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence complementary to at least a portion ofa sequence from an odd numbered sequence ranging from SEQ ID NO: 1 toSEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, said portioncomprising at least 10 nucleotides.

The present invention provides a method of producing a polypeptidecomprising a sequence from an even numbered sequence ranging from SEQ IDNO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, or a homologor fragment thereof. The method comprising the steps of introducing arecombinant expression vector that includes a nucleotide sequence thatencodes the polypeptide into a compatible host cell, growing the hostcell under conditions for expression of the polypeptide and recoveringthe polypeptide.

The present invention provides an isolated antibody which binds to anepitope on a polypeptide comprising a sequence from an even numberedsequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 andSEQ ID NO: 192, or a homolog or fragment thereof.

The present invention provides an method of inducing an immune responsein a mammal against a polypeptide comprising a sequence from an evennumbered sequence ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO:186 and SEQ ID NO: 192, or a homolog or fragment thereof. The methodcomprises administering to a mammal an amount of the polypeptidesufficient to induce said immune response.

The present invention provides a method for identifying a compound whichbinds nGPCR-x. The method comprises the steps of: contacting nGPCR-xwith a compound and determining whether the compound binds nGPCR-x.

The present invention provides a method for identifying a compound whichbinds a nucleic acid molecule encoding nGPCR-x. The method comprises thesteps of contacting said nucleic acid molecule encoding nGPCR-x with acompound and determining whether said compound binds said nucleic acidmolecule.

The present invention provides a method for identifying a compound whichmodulates the activity of nGPCR-x. The method comprises the steps ofcontacting nGPCR-x with a compound and determining whether nGPCR-xactivity has been modulated.

The present invention provides a method of identifying an animal homologof nGPCR-x. The method comprises the steps screening a nucleic aciddatabase of the animal with an odd numbered sequence ranging from SEQ IDNO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or a portionthereof and determining whether a portion of said library or database ishomologous to said odd numbered sequence ranging from SEQ ID NO: 1 toSEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, or portion thereof.

The present invention provides a method of identifying an animal homologof nGPCR-x. The methods comprises the steps screening a nucleic acidlibrary of the animal with a nucleic acid molecule having an oddnumbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93,SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof; and determiningwhether a portion of said library or database is homologous to said oddnumbered nucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93,SEQ ID NO: 185 and SEQ ID NO: 191, or a portion thereof.

Another aspect of the present invention relates to methods of screeninga human subject to diagnose a disorder affecting the brain or geneticpredisposition therefor. The methods comprise the steps of assayingnucleic acid of a human subject to determine a presence or an absence ofa mutation altering an amino acid sequence, expression, or biologicalactivity of at least one nGPCR that is expressed in the brain. The nGPCRcomprise an amino acid sequence selected from the group consisting of:SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, and SEQ ID NO:94, and allelicvariants thereof. A diagnosis of the disorder or predisposition is madefrom the presence or absence of the mutation. The presence of a mutationaltering the amino acid sequence, expression, or biological activity ofthe nGPCR in the nucleic acid correlates with an increased risk ofdeveloping the disorder.

The present invention further relates to methods of screening for anGPCR-40 or nGPCR-54 hereditary schizophrenia genotype in a humanpatient. The methods comprise the steps of providing a biological samplecomprising nucleic acid from the patient, in which the nucleic acidincludes sequences corresponding to alleles of nGPCR-40 or nGPCR-54. Thepresence of one or more mutations in the nGPCR-40 allele or the nGPCR-54allele is detected indicative of a hereditary schizophrenia genotype.

The present invention provides kits for screening a human subject todiagnose schizophrenia or a genetic predisposition therefor. The kitsinclude an oligonucleotide useful as a probe for identifyingpolymorphisms in a human nGPCR-40 gene or a human nGPCR-54 gene. Theoligonucleotide comprises 6-50 nucleotides in a sequence that isidentical or complementary to a sequence of a wild type human nGPCR-40or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 coding sequence,except for one sequence difference selected from the group consisting ofa nucleotide addition, a nucleotide deletion, or nucleotidesubstitution. The kit also includes a media packaged with theoligonucleotide. The media contains information for identifyingpolymorphisms that correlate with schizophrenia or a geneticpredisposition therefor, the polymorphisms being identifiable using theoligonucleotide as a probe.

The present invention further relates to methods of identifying nGPCRallelic variants that correlates with mental disorders. The methodscomprise the steps of providing biological samples that comprise nucleicacid from a human patient diagnosed with a mental disorder, or from thepatient's genetic progenitors or progeny, and detecting in the nucleicacid the presence of one or more mutations in an nGPCR that is expressedin the brain. The nGPCR comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:74, SEQ ID NO: 186, SEQ ID NO:78, SEQID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:90, andSEQ ID NO:94, and allelic variants thereof. The nucleic acid includessequences corresponding to the gene or genes encoding nGPCR. The one ormore mutations detected indicate an allelic variant that correlates witha mental disorder.

The present invention further relates to purified polynucleotidescomprising nucleotide sequences encoding alleles of nGPCR-40 or nGPCR-54from a human with schizophrenia. The polynucleotide hybridizes to thecomplement of SEQ ID NO:83 or of SEQ ID NO:85 under the followinghybridization conditions: (a) hybridization for 16 hours at 42° C. in ahybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10%dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in awash solution comprising 0.1×SSC and 1% SDS. The polynucleotide thatencodes nGPCR-40 or nGPCR-54 amino acid sequence of the human differsfrom SEQ ID NO:84 or SEQ ID NO:86 by at least one residue.

The present invention also provides methods for identifying a modulatorof biological activity of nGPCR-40 or nGPCR-54 comprising the steps ofcontacting a cell that expresses nGPCR-40 or nGPCR-54 in the presenceand in the absence of a putative modulator compound and measuringnGPCR-40 or nGPCR-54 biological activity in the cell. The decreased orincreased nGPCR-40 or nGPCR-54 biological activity in the presenceversus absence of the putative modulator is indicative of a modulator ofbiological activity.

The present invention further provides methods to identify compoundsuseful for the treatment of schizophrenia. The methods comprise thesteps of contacting a composition comprising nGPCR-40 with a compoundsuspected of binding nGPCR-40 or contacting a composition comprisingnGPCR-54 with a compound suspected of binding nGPCR-54. The bindingbetween nGPCR-40 and the compound suspected of binding nGPCR-40 orbetween nGPCR-54 and the compound suspected of binding nGPCR-54 isdetected. Compounds identified as binding nGPCR-40 or nGPCR-54 arecandidate compounds useful for the treatment of schizophrenia.

The present invention further provides methods for identifying acompound useful as a modulator of binding between nGPCR-40 and a bindingpartner of nGPCR-40 or between nGPCR-54 and a binding partner ofnGPCR-54. The methods comprise the steps of contacting the bindingpartner and a composition comprising nGPCR-40 or nGPCR-54 in thepresence and in the absence of a putative modulator compound anddetecting binding between the binding partner and nGPCR-40 or nGPCR-54.Decreased or increased binding between the binding partner and nGPCR-40or nGPCR-54 in the presence of the putative modulator, as compared tobinding in the absence of the putative modulator is indicative amodulator compound useful for the treatment of schizophrenia.

Another aspect of the present invention relates to methods of purifyinga G protein from a sample containing a G protein. The methods comprisethe steps of contacting the sample with an nGPCR for a time sufficientto allow the G protein to form a complex with the nGPCR; isolating thecomplex from remaining components of the sample; maintaining the complexunder conditions which result in dissociation of the G protein from thenGPCR; and isolating said G protein from the nGPCR.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

Various definitions are made throughout this document. Most words havethe meaning that would be attributed to those words by one skilled inthe art. Words specifically defined either below or elsewhere in thisdocument have the meaning provided in the context of the presentinvention as a whole and as are typically understood by those skilled inthe art.

“Synthesized” as used herein and understood in the art, refers topolynucleotides produced by purely chemical, as opposed to enzymatic,methods. “Wholly” synthesized DNA sequences are therefore producedentirely by chemical means, and “partially” synthesized DNAs embracethose wherein only portions of the resulting DNA were produced bychemical means.

By the term “region” is meant a physically contiguous portion of theprimary structure of a biomolecule. In the case of proteins, a region isdefined by a contiguous portion of the amino acid sequence of thatprotein.

The term “domain” is herein defined as referring to a structural part ofa biomolecule that contributes to a known or suspected function of thebiomolecule. Domains may be co-extensive with regions or portionsthereof; domains may also incorporate a portion of a biomolecule that isdistinct from a particular region, in addition to all or part of thatregion. Examples of GPCR protein domains include, but are not limitedto, the extracellular (i.e., N-terminal), transmembrane and cytoplasmic(i.e., C-terminal) domains, which are co-extensive with like-namedregions of GPCRS; each of the seven transmembrane segments of a GPCR;and each of the loop segments (both extracellular and intracellularloops) connecting adjacent transmembrane segments.

As used herein, the term “activity” refers to a variety of measurableindicia suggesting or revealing binding, either direct or indirect;affecting a response, i.e. having a measurable affect in response tosome exposure or stimulus, including, for example, the affinity of acompound for directly binding a polypeptide or polynucleotide of theinvention, or, for example, measurement of amounts of upstream ordownstream proteins or other similar functions after some stimulus orevent.

Unless indicated otherwise, as used herein, the abbreviation in lowercase (gpcr) refers to a gene, cDNA, RNA or nucleic acid sequence, whilethe upper case version (GPCR) refers to a protein, polypeptide, peptide,oligopeptide, or amino acid sequence. The term “nGPCR-x” refers to anyof the nGPCRs taught herein, while specific reference to a nGPCR (forexample nGPCR-5) refers only to that specific nGPCR.

As used herein, the term “antibody” is meant to refer to complete,intact antibodies, and Fab, Fab′, F(ab)₂, and other fragments thereof.Complete, intact antibodies include monoclonal antibodies such as murinemonoclonal antibodies, chimeric antibodies and humanized antibodies.

As used herein, the term “binding” means the physical or chemicalinteraction between two proteins or compounds or associated proteins orcompounds or combinations thereof. Binding includes ionic, nononic,Hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Thephysical interaction, the binding, can be either direct or indirect,indirect being through or due to the effects of another protein orcompound. Direct binding refers to interactions that do not take placethrough or due to the effect of another protein or compound but insteadare without other substantial chemical intermediates. Binding may bedetected in many different manners. As a non-limiting example, thephysical binding interaction between a nGPCR-x of the invention and acompound can be detected using a labeled compound. Alternatively,functional evidence of binding can be detected using, for example, acell transfected with and expressing a nGPCR-x of the invention. Bindingof the transfected cell to a ligand of the nGPCR that was transfectedinto the cell provides functional evidence of binding. Other methods ofdetecting binding are well-known to those of skill in the art.

As used herein, the term “compound” means any identifiable chemical ormolecule, including, but not limited to, small molecule, peptide,protein, sugar, nucleotide, or nucleic acid, and such compound can benatural or synthetic.

As used herein, the term “complementary” refers to Watson-Crickbasepairing between nucleotide units of a nucleic acid molecule.

As used herein, the term “contacting” means bringing together, eitherdirectly or indirectly, a compound into physical proximity to apolypeptide or polynucleotide of the invention. The polypeptide orpolynucleotide can be in any number of buffers, salts, solutions etc.Contacting includes, for example, placing the compound into a beaker,microtiter plate, cell culture flask, or a microarray, such as a genechip, or the like, which contains the nucleic acid molecule, orpolypeptide encoding the nGPCR or fragment thereof.

As used herein, the phrase “homologous nucleotide sequence,” or“homologous amino acid sequence,” or variations thereof, refers tosequences characterized by a homology, at the nucleotide level or aminoacid level, of at least the specified percentage. Homologous nucleotidesequences include those sequences coding for isoforms of proteins. Suchisoforms can be expressed in different tissues of the same organism as aresult of, for example, alternative splicing of RNA. Alternatively,isoforms can be encoded by different genes. Homologous nucleotidesequences include nucleotide sequences encoding for a protein of aspecies other than humans, including, but not limited to, mammals.Homologous nucleotide sequences also include, but are not limited to,naturally occurring allelic variations and mutations of the nucleotidesequences set forth herein. A homologous nucleotide sequence does not,however, include the nucleotide sequence encoding other known GPCRs.Homologous amino acid sequences include those amino acid sequences whichcontain conservative amino acid substitutions and which polypeptideshave the same binding and/or activity. A homologous amino acid sequencedoes not, however, include the amino acid sequence encoding other knownGPCRs. Percent homology can be determined by, for example, the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, Madison Wis.), usingthe default settings, which uses the algorithm of Smith and Waterman(Adv. Appl. Math., 1981, 2, 482 489, which is incorporated herein byreference in its entirety).

As used herein, the term “isolated” nucleic acid molecule refers to anucleic acid molecule (DNA or RNA) that has been removed from its nativeenvironment. Examples of isolated nucleic acid molecules include, butare not limited to, recombinant DNA molecules contained in a vector,recombinant DNA molecules maintained in a heterologous host cell,partially or substantially purified nucleic acid molecules, andsynthetic DNA or RNA molecules.

As used herein, the terms “modulates” or “modifies” means an increase ordecrease in the amount, quality, or effect of a particular activity orprotein.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues which has a sufficient number of bases to be used ina polymerase chain reaction (PCR). This short sequence is based on (ordesigned from) a genomic or cDNA sequence and is used to amplify,confirm, or reveal the presence of an identical, similar orcomplementary DNA or RNA in a particular cell or tissue.Oligonucleotides comprise portions of a DNA sequence having at leastabout 10 nucleotides and as many as about 50 nucleotides, preferablyabout 15 to 30 nucleotides. They are chemically synthesized and may beused as probes.

As used herein, the term “probe” refers to nucleic acid sequences ofvariable length, preferably between at least about 10 and as many asabout 6,000 nucleotides, depending on use. They are used in thedetection of identical, similar, or complementary nucleic acidsequences. Longer length probes are usually obtained from a natural orrecombinant source, are highly specific and much slower to hybridizethan oligomers. They may be single- or double-stranded and carefullydesigned to have specificity in PCR, hybridization membrane based, orELISA-like technologies.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops an abnormal condition.

The term “treating” refers to having a therapeutic effect and at leastpartially alleviating or abrogating an abnormal condition in theorganism.

The term “therapeutic effect” refers to the inhibition or activationfactors causing or contributing to the abnormal condition. A therapeuticeffect relieves to some extent one or more of the symptoms of theabnormal condition. In reference to the treatment of abnormalconditions, a therapeutic effect can refer to one or more of thefollowing: (a) an increase in the proliferation, growth, and/ordifferentiation of cells; (b) inhibition (i.e., slowing or stopping) ofcell death; (c) inhibition of degeneration; (d) relieving to some extentone or more of the symptoms associated with the abnormal condition; and(e) enhancing the function of the affected population of cells.Compounds demonstrating efficacy against abnormal conditions can beidentified as described herein.

The term “abnormal condition” refers to a function in the cells ortissues of an organism that deviates from their normal functions in thatorganism. An abnormal condition can relate to cell proliferation, celldifferentiation, cell signaling, or cell survival. An abnormal conditionmay also include obesity, diabetic complications such as retinaldegeneration, and irregularities in glucose uptake and metabolism, andfatty acid uptake and metabolism.

Abnormal cell proliferative conditions include cancers such as fibroticand mesangial disorders, abnormal angiogenesis and vasculogenesis, woundhealing, psoriasis, diabetes mellitus, and inflammation.

Abnormal differentiation conditions include, but are not limited to,neurodegenerative disorders, slow wound healing rates, and slow tissuegrafting healing rates. Abnormal cell signaling conditions include, butare not limited to, psychiatric disorders involving excessneurotransmitter activity.

Abnormal cell survival conditions may also relate to conditions in whichprogrammed cell death (apoptosis) pathways are activated or abrogated. Anumber of protein kinases are associated with the apoptosis pathways.Aberrations in the function of any one of the protein kinases could leadto cell immortality or premature cell death.

The term “administering” relates to a method of incorporating a compoundinto cells or tissues of an organism. The abnormal condition can beprevented or treated when the cells or tissues of the organism existwithin the organism or outside of the organism. Cells existing outsidethe organism can be maintained or grown in cell culture dishes. Forcells harbored within the organism, many techniques exist in the art toadminister compounds, including (but not limited to) oral, parenteral,dermal, injection, and aerosol applications. For cells outside of theorganism, multiple techniques exist in the art to administer thecompounds, including (but not limited to) cell microinjectiontechniques, transformation techniques and carrier techniques.

The abnormal condition can also be prevented or treated by administeringa compound to a group of cells having an aberration in a signaltransduction pathway to an organism. The effect of administering acompound on organism function can then be monitored. The organism ispreferably a mouse, rat, rabbit, guinea pig or goat, more preferably amonkey or ape, and most preferably a human.

By “amplification” it is meant increased numbers of DNA or RNA in a cellcompared with normal cells. “Amplification” as it refers to RNA can bethe detectable presence of RNA in cells, since in some normal cellsthere is no basal expression of RNA. In other normal cells, a basallevel of expression exists, therefore in these cases amplification isthe detection of at least 1 to 2-fold, and preferably more, compared tothe basal level.

As used herein, the phrase “stringent hybridization conditions” or“stringent conditions” refers to conditions under which a probe, primer,or oligonucleotide will hybridize to its target sequence, but to noother sequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present in excess, at T_(m), 50% of theprobes are occupied at equilibrium. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes,primers or oligonucleotides (e.g. 10 to 50 nucleotides) and at leastabout 60° C. for longer probes, primers or oligonucleotides. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide.

The amino acid sequences are presented in the amino to carboxydirection, from left to right. The amino and carboxy groups are notpresented in the sequence. The nucleotide sequences are presented bysingle strand only, in the 5′ to 3′ direction, from left to right.Nucleotides and amino acids are represented in the manner recommended bythe IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids)by three letters code.

Polynucleotides

The present invention provides purified and isolated polynucleotides(e.g., DNA sequences and RNA transcripts, both sense and complementaryantisense strands, both single- and double-stranded, including splicevariants thereof) that encode unknown G protein-coupled receptorsheretofore termed novel GPCRs, or nGPCRs. These genes are describedherein and designated herein collectively as nGPCR-x (where x is 1, 3,4, 5, 9, 11, 12, 14, 15, 18, 16, 17, 20, 21, 22, 24, 27, 28, 31, 32, 33,34, 35, 36, 37, 38, 40, 41, 53, 54, 55, 56, 57, 58, 59, or 60). That is,these genes are described herein and designated herein as nGPCR-1 (alsoreferred to as beGPCR-1), nGPCR-3 (also referred to as beGPCR-3),nGPCR-4 (also referred to as beGPCR-4), nGPCR-5 (also referred to asbeGPCR-5 and TL-GPCR-5), nGPCR-9 (also referred to as beGPCR-9),nGPCR-11 (also referred to as beGPCR-11), nGPCR-12 (also referred to asbeGPCR-12), nGPCR-14 (also referred to as beGPCR-14), nGPCR-15 (alsoreferred to as beGPCR-15), nGPCR-18 (also referred to as beGPCR-18),nGPCR-16 (also referred to as beGPCR-16), nGPCR-17 (also referred to asbeGPCR-17), nGPCR-20 (also referred to as beGPCR-20), nGPCR-21 (alsoreferred to as beGPCR-21), nGPCR-22 (also referred to as beGPCR-22),nGPCR-24 (also referred to as beGPCR-24), nGPCR-27 (also referred to asbeGPCR-27), nGPCR-28 (also referred to as beGPCR-28), nGPCR-31 (alsoreferred to as beGPCR-31), nGPCR-32 (also referred to as beGPCR-32),nGPCR-33 (also referred to as beGPCR-33), nGPCR-34 (also referred to asbeGPCR-34), nGPCR-35 (also referred to as beGPCR-35), nGPCR-36 (alsoreferred to as beGPCR-36), nGPCR-37 (also referred to as beGPCR-37),nGPCR-38 (also referred to as beGPCR-38), nGPCR-40 (also referred to asbeGPCR-40), nGPCR-1 (also referred to as beGPCR-41), nGPCR-53, nGPCR-54,nGPCR-55, nGPCR-56, nGPCR-57, nGPCR-58, nGPCR-59, and nGPCR-60. Table 1below identifies the novel gene sequence nGPCR-x designation, the SEQ IDNO: of the gene sequence, the SEQ ID NO: of the polypeptide hereby, andthe U.S. Provisional Application in which the gene sequence has beenTABLE 1 Nucleotide Amino acid Sequence Sequence (SEQ ID (SEQ IDOriginally nGPCR NO:) NO:) filed in: 1 1 2 A 1 73 74 E 3 3 4 A 3 185 186P 4 5 6 A 5 7 8 A 5 75 76 F 9 9 10 A 9 77 78 G 11 11 12 A 11 79 80 H 1213 14 A 14 15 16 A 14 191 192 herein 15 17 18 A 18 19 20 A 16 21 22 B 1681 82 I 17 23 24 B 20 25 26 B 21 27 28 B 22 29 30 B 24 31 32 B 27 33 34B 28 35 36 B 31 37 38 B 32 39 40 B 33 41 42 C 34 43 44 C 35 45 46 C 3647 48 C 37 49 50 C 38 51 52 C 40 53 54 C 40 83 84 J 41 55 56 C 53 57 58D 54 59 60 D 54 85 86 K 55 61 62 D 56 63 64 D 56 87 88 L 56 89 90 M 5765 66 D 58 67 68 D 58 91 92 N 58 93 94 O 59 69 70 D 60 71 72 DLegendA = Ser. No. 60/165,838B = Ser. No. 60/166,701C = Ser. No. 60/166,678D = Ser. No. 60/173,396E = Ser. No. 60/184,129F = Ser. No. 60/188,114G = Ser. No. 60/185,421H = Ser. No. 60/186,811I = Ser. No. 60/186,530J = Ser. No. 60/207,094K = Ser. No. 60/203,111L = Ser. No. 60/190,310M = Ser. No. 60/201,190N = Ser. No. 60/185554O = Ser. No. 60/190,800P = Ser. No. 60/198,568

When a specific nGPCR is identified (for example nGPCR-5), it isunderstood that only that specific nGPCR is being referred to.

As described in Example 4 below, the genes encoding nGPCR-1 (nucleicacid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequence SEQ IDNO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9, SEQ IDNO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11(nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequenceSEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO:21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82),nGPCR-40 (nucleic acids sequence SEQ ID NO:53, SEQ ID NO:83, amino acidsequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequenceSEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO:86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ IDNO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90),nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ IDNO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94)and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, aminoacid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in braintissue indicating that these n-GPCR-x proteins are neuroreceptors.

The invention provides purified and isolated polynucleotides (e.g.,cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whethersingle or double-stranded) that comprise a nucleotide sequence encodingthe amino acid sequence of the polypeptides of the invention. Suchpolynucleotides are useful for recombinantly expressing the receptor andalso for detecting expression of the receptor in cells (e.g., usingNorthern hybridization and in situ hybridization assays). Suchpolynucleotides also are useful in the design of antisense and othermolecules for the suppression of the expression of nGPCR-x in a culturedcell, a tissue, or an animal; for therapeutic purposes; or to provide amodel for diseases or conditions characterized by aberrant nGPCR-xexpression. Specifically excluded from the definition of polynucleotidesof the invention are entire isolated, non-recombinant native chromosomesof host cells. A preferred polynucleotide has the sequence of thesequence set forth in odd numbered sequences ranging from SEQ ID NO: 1to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191, which correspond tonaturally occurring nGPCR-x sequences. It will be appreciated thatnumerous other polynucleotide sequences exist that also encode nGPCR-xhaving the sequence set forth in even numbered sequences ranging fromSEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192, due tothe well-known degeneracy of the universal genetic code.

The invention also provides a purified and isolated polynucleotidecomprising a nucleotide sequence that encodes a mammalian polypeptide,wherein the polynucleotide hybridizes to a polynucleotide having thesequence set forth in odd numbered sequences ranging from SEQ ID NO: 1to SEQ ID NO: 93, SEQ ID NO: 185, and SEQ ID NO:191, or the non-codingstrand complementary thereto, under the following hybridizationconditions:

-   -   (a) hybridization for 16 hours at 42° C. in a hybridization        solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran        sulfate; and    -   (b) washing 2 times for 30 minutes each at 60° C. in a wash        solution comprising 0.1% SSC, 1% SDS. Polynucleotides that        encode a human allelic variant are highly preferred.

The present invention relates to molecules which comprise the genesequences that encode the nGPCRs; constructs and recombinant host cellsincorporating the gene sequences; the novel GPCR polypeptides encoded bythe gene sequences; antibodies to the polypeptides and homologs; kitsemploying the polynucleotides and polypeptides, and methods of makingand using all of the foregoing. In addition, the present inventionrelates to homologs of the gene sequences and of the polypeptides andmethods of making and using the same.

Genomic DNA of the invention comprises the protein-coding region for apolypeptide of the invention and is also intended to include allelicvariants thereof. It is widely understood that, for many genes, genomicDNA is transcribed into RNA transcripts that undergo one or moresplicing events wherein intron (i.e., non-coding regions) of thetranscripts are removed, or “spliced out.” RNA transcripts that can bespliced by alternative mechanisms, and therefore be subject to removalof different RNA sequences but still encode a nGPCR-x polypeptide, arereferred to in the art as splice variants which are embraced by theinvention. Splice variants comprehended by the invention therefore areencoded by the same original genomic DNA sequences but arise fromdistinct mRNA transcripts Allelic variants are modified forms of awild-type gene sequence, the modification resulting from recombinationduring chromosomal segregation or exposure to conditions which give riseto genetic mutation. Allelic variants, like wild type genes, arenaturally occurring sequences (as opposed to non-naturally occurringvariants that arise from in vitro manipulation).

The invention also comprehends cDNA that is obtained through reversetranscription of an RNA polynucleotide encoding nGPCR-x (conventionallyfollowed by second strand synthesis of a complementary strand to providea double-stranded DNA).

Preferred DNA sequences encoding human nGPCR-x polypeptides are set outin odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93,SEQ ID NO: 185 and SEQ ID NO: 191. A preferred DNA of the inventioncomprises a double stranded molecule along with the complementarymolecule (the “non-coding strand” or “complement”) having a sequenceunambiguously deducible from the coding strand according to Watson-Crickbase-pairing rules for DNA. Also preferred are other polynucleotidesencoding the nGPCR-x polypeptide of even numbered sequences ranging fromSEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192 whichdiffer in sequence from the polynucleotides of odd numbered sequencesranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ IDNO: 192, by virtue of the well-known degeneracy of the universal nucleargenetic code.

In a preferred embodiment, the isolated nucleic acid molecule comprisesa nucleotide sequence which encodes a fragment of polypeptide comprisinga sequence of SEQ ID NO: 192. The fragment of the polypeptide comprisinga sequence of SEQ ID NO: 192 comprises at least one or more amino acidresidues from one or more of the following regions of SEQ ID NO: 192:amino acid residues 1 to 42 of SEQ ID NO: 192; amino acid residues 68 to77 of SEQ ID NO: 192; amino acid residues 185 to 197 of SEQ ID NO: 192;or amino acid residues 293 to 513 of SEQ ID NO: 192.

In a preferred embodiment, the isolated nucleic acid comprises anucleotide sequence of SEQ ID NO: 191, and fragments thereof, thatencode a polypeptide having a sequence of SEQ ID NO: 192, or fragmentsthereof. The fragment of the nucleotide sequence of SEQ ID NO: 191comprises at least one or more nucleotides from one or more of thefollowing regions of SEQ ID NO: 191: nucleotides 1 to 193 of SEQ ID NO:191; nucleotides 612 to 644 of SEQ ID NO:191; nucleotides 697 to 706 ofSEQ ID NO:191; nucleotides 1011 to 1049 of SEQ ID NO: 191; nucleotides1051 to 1057 of SEQ ID NO:191; nucleotides 1090 to 1096 of SEQ IDNO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.

The invention further embraces other species, preferably mammalian,homologs of the human nGPCR-x DNA. Species homologs, sometimes referredto as “orthologs,” in general, share at least 35%, at least 40%, atleast 45%, at least 50%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% homology with human DNA of the invention.Generally, percent sequence “homology” with respect to polynucleotidesof the invention may be calculated as the percentage of nucleotide basesin the candidate sequence that are identical to nucleotides in thenGPCR-x sequence set forth in odd numbered sequences ranging from SEQ IDNO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO: 191, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity.

Polynucleotides of the invention permit identification and isolation ofpolynucleotides encoding related nGPCR-x polypeptides, such as humanallelic variants and species homologs, by well-known techniquesincluding Southern and/or Northern hybridization, and polymerase chainreaction (PCR). Examples of related polynucleotides include human andnon-human genomic sequences, including allelic variants, as well aspolynucleotides encoding polypeptides homologous to nGPCR-x andstructurally related polypeptides sharing one or more biological,immunological, and/or physical properties of nGPCR-x. Non-human speciesgenes encoding proteins homologous to nGPCR-x can also be identified bySouthern and/or PCR analysis and are useful in animal models for nGPCR-xdisorders. Knowledge of the sequence of a human nGPCR-x DNA also makespossible through use of Southern hybridization or polymerase chainreaction (PCR) the identification of genomic DNA sequences encodingnGPCR-x expression control regulatory sequences such as promoters,operators, enhancers, repressors, and the like. Polynucleotides of theinvention are also useful in hybridization assays to detect the capacityof cells to express nGPCR-x. Polynucleotides of the invention may alsoprovide a basis for diagnostic methods useful for identifying a geneticalteration(s) in a nGPCR-x locus that underlies a disease state orstates, which information is useful both for diagnosis and for selectionof therapeutic strategies.

According to the present invention, the nGPCR-x nucleotide sequencesdisclosed herein may be used to identify homologs of the nGPCR-x, inother animals, including but not limited to humans and other mammals,and invertebrates. Any of the nucleotide sequences disclosed herein, orany portion thereof, can be used, for example, as probes to screendatabases or nucleic acid libraries, such as, for example, genomic orcDNA libraries, to identify homologs, using screening procedures wellknown to those skilled in the art. Accordingly, homologs having at least50%, more preferably at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, more preferablyat least 95%, and most preferably at least 100% homology with nGPCR-xsequences can be identified.

The disclosure herein of full-length polynucleotides encoding nGPCR-xpolypeptides makes readily available to the worker of ordinary skill inthe art every possible fragment of the full-length polynucleotide.

One preferred embodiment of the present invention provides an isolatednucleic acid molecule comprising a sequence homologous to odd numberedsequences selected from the group consisting of SEQ ID NO: 1 to SEQ IDNO:93, SEQ ID NO: 185 and SEQ ID NO: 191, and fragments thereof. Anotherpreferred embodiment provides an isolated nucleic acid moleculecomprising a sequence selected from the group of odd numbered sequencesconsisting of SEQ ID NO: 1 to SEQ ID NO:93, SEQ ID NO: 185 and SEQ IDNO: 191, and fragments thereof.

As used in the present invention, fragments of nGPCR-x-encodingpolynucleotides comprise at least 10, and preferably at least 12, 14,16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotideencoding nGPCR-x. Preferably, fragment polynucleotides of the inventioncomprise sequences unique to the nGPCR-x-encoding polynucleotidesequence, and therefore hybridize under highly stringent or moderatelystringent conditions only (i.e., “specifically”) to polynucleotidesencoding nGPCR-x (or fragments thereof). Polynucleotide fragments ofgenomic sequences of the invention comprise not only sequences unique tothe coding region, but also include fragments of the full-lengthsequence derived from introns, regulatory regions, and/or othernon-translated sequences. Sequences unique to polynucleotides of theinvention are recognizable through sequence comparison to other knownpolynucleotides, and can be identified through use of alignment programsroutinely utilized in the art, e.g., those made available in publicsequence databases. Such sequences also are recognizable from Southernhybridization analyses to determine the number of fragments of genomicDNA to which a polynucleotide will hybridize. Polynucleotides of theinvention can be labeled in a manner that permits their detection,including radioactive, fluorescent, and enzymatic labeling.

Fragment polynucleotides are particularly useful as probes for detectionof full-length or fragments of nGPCR-x polynucleotides. One or morepolynucleotides can be included in kits that are used to detect thepresence of a polynucleotide encoding nGPCR-x, or used to detectvariations in a polynucleotide sequence encoding nGPCR-x.

The invention also embraces DNAs encoding nGPCR-x polypeptides thathybridize under moderately stringent or high stringency conditions tothe non-coding strand, or complement, of the polynucleotides set forthin odd numbered sequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93,SEQ ID NO: 185 and SEQ ID NO:192.

Exemplary highly stringent hybridization conditions are as follows:hybridization at 42° C. in a hybridization solution comprising 50%formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS.It is understood in the art that conditions of equivalent stringency canbe achieved through variation of temperature and buffer, or saltconcentration as described Ausubel et al. (Eds.), Protocols in MolecularBiology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications inhybridization conditions can be empirically determined or preciselycalculated based on the length and the percentage of guanosine/cytosine(GC) base pairing of the probe. The hybridization conditions can becalculated as described in Sambrook, et al., (Eds.), Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold SpringHarbor, N.Y. (1989), pp. 9.47 to 9.51.

With the knowledge of the nucleotide sequence information disclosed inthe present invention, one skilled in the art can identify and obtainnucleotide sequences which encode nGPCR-x from different sources (i.e.,different tissues or different organisms) through a variety of meanswell known to the skilled artisan and as disclosed by, for example,Sambrook et al., “Molecular cloning: a laboratory manual”, SecondEdition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (19.89),which is incorporated herein by reference in its entirety.

For example, DNA that encodes nGPCR-x may be obtained by screening ofmRNA, cDNA, or genomic DNA with oligonucleotide probes generated fromthe nGPCR-x gene sequence information provided herein. Probes may belabeled with a detectable group, such as a fluorescent group, aradioactive atom or a chemiluminescent group in accordance withprocedures known to the skilled artisan and used in conventionalhybridization assays, as described by, for example, Sambrook et al.

A nucleic acid molecule comprising any of the nGPCR-x nucleotidesequences described above can alternatively be synthesized by use of thepolymerase chain reaction (PCR) procedure, with the PCR oligonucleotideprimers produced from the nucleotide sequences provided herein. See U.S.Pat. No. 4,683,195 to Mulliset al. and U.S. Pat. No. 4,683,202 toMullis. The PCR reaction provides a method for selectively increasingthe concentration of a particular nucleic acid sequence even when thatsequence has not been previously purified and is present only in asingle copy in a particular sample. The method can be used to amplifyeither single- or double-stranded DNA. The essence of the methodinvolves the use of two oligonucleotide probes to serve as primers forthe template dependent, polymerase mediated replication of a desirednucleic acid molecule.

A wide variety of alternative cloning and in vitro amplificationmethodologies are well known to those skilled in the art Examples ofthese techniques are found in, for example, Berger et al., Guide toMolecular Cloning Techniques, Methods in Enzymology 152, Academic Press,Inc., San Diego, Calif. (Berger), which is incorporated herein byreference in its entirety.

Automated sequencing methods can be used to obtain or verify thenucleotide sequence of nGPCR-x. The nGPCR-x nucleotide sequences of thepresent invention are believed to be 100% accurate. However, as is knownin the art, nucleotide sequence obtained by automated methods maycontain some errors. Nucleotide sequences determined by automation aretypically at least about 90%, more typically at least about 95% to atleast about 99.9% identical to the actual nucleotide sequence of a givennucleic acid molecule. The actual sequence may be more preciselydetermined using manual sequencing methods, which are well known in theart. An error in a sequence which results in an insertion or deletion ofone or more nucleotides may result in a frame shift in translation suchthat the predicted amino acid sequence will differ from that which wouldbe predicted from the actual nucleotide sequence of the nucleic acidmolecule, starting at the point of the mutation.

The nucleic acid molecules of the present invention, and fragmentsderived therefrom, are useful for screening for restriction fragmentlength polymorphism (RFLP) associated with certain disorders, as well asfor genetic mapping.

The polynucleotide sequence information provided by the invention makespossible large-scale expression of the encoded polypeptide by techniqueswell known and routinely practiced in the art.

Vectors

Another aspect of the present invention is directed to vectors, orrecombinant expression vectors, comprising any of the nucleic acidmolecules described above. Vectors are used herein either to amplify DNAor RNA encoding nGPCR-x and/or to express DNA which encodes nGPCR-x.Preferred vectors include, but are not limited to, plasmids, phages,cosmids, episomes, viral particles or viruses, and integratable DNAfragments (i.e., fragments integratable into the host genome byhomologous recombination). Preferred viral particles include, but arenot limited to, adenoviruses, baculoviruses, parvoviruses,herpesviruses, poxviruses, adeno-associated viruses, Semliki Forestviruses, vaccinia viruses, and retroviruses. Preferred expressionvectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL(Pharmacia Biotech). Other expression vectors include, but are notlimited to, pSPORT™ vectors, pGEM™ vectors (Promega), pPROEX vectors™(LTI, Bethesda, Md.), Bluescript™ vectors (Stratagene), pQE™ vectors(Qiagen), pSE420™ (Invitrogen), and pYES2™ (Invitrogen).

Expression constructs preferably comprise GPCR-x-encodingpolynucleotides operatively linked to an endogenous or exogenousexpression control DNA sequence and a transcription terminator.Expression control DNA sequences include promoters, enhancers,operators, and regulatory element binding sites generally, and oretypically selected based on the expression systems in which theexpression construct is to be utilized. Preferred promoter and enhancersequences are generally selected for the ability to increase geneexpression, while operator sequences are generally selected for theability to regulate gene expression. Expression constructs of theinvention may also include sequences encoding one or more selectablemarkers that permit identification of host cells bearing the construct.Expression constructs may also include sequences that facilitate, andpreferably promote, homologous recombination in a host cell. Preferredconstructs of the invention also include sequences necessary forreplication in a host cell.

Expression constructs are preferably utilized for production of anencoded protein, but may also be utilized simply to amplify anGPCR-x-encoding polynucleotide sequence. In preferred embodiments, thevector is an expression vector wherein the polynucleotide of theinvention is operatively linked to a polynucleotide comprising anexpression control sequence. Autonomously replicating recombinantexpression constructs such as plasmid and viral DNA vectorsincorporating polynucleotides of the invention are also provided.Preferred expression vectors are replicable DNA constructs in which aDNA sequence encoding nGPCR-x is operably linked or connected tosuitable control sequences capable of effecting the expression of thenGPCR-x in a suitable host DNA regions are operably linked or connectedwhen they are functionally related to each other. For example, apromoter is operably linked or connected to a coding sequence if itcontrols the transcription of the sequence. Amplification vectors do notrequire expression control domains, but rather need only the ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants. The needfor control sequences in the expression vector will vary depending uponthe host selected and the transformation method chosen. Generally,control sequences include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding and sequences which control the termination oftranscription and translation.

Preferred vectors preferably contain a promoter that is recognized bythe host organism. The promoter sequences of the present invention maybe prokaryotic, eukaryotic or viral. Examples of suitable prokaryoticsequences include the P_(R) and P_(L) promoters of bacteriophage lambda(The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1973), which is incorporated herein byreference in its entirety; Lambda II, Hendrix, R. W., Ed., Cold SpringHabor Press, Cold Spring Harbor, N.Y. (1980), which is incorporatedherein by reference in its entirety); the trp, recA, heat shock, andlacZ promoters of E. coli and the SV40 early promoter (Benoist et al.Nature, 1981, 290, 304-310, which is incorporated herein by reference inits entirety). Additional promoters include, but are not limited to,mouse mammary tumor virus, long terminal repeat of humanimmunodeficiency virus, maloney virus, cytomegalovirus immediate earlypromoter, Epstein Barr virus, Rous sarcoma virus, human actin, humanmyosin, human hemoglobin, human muscle creatine, and humanmetalothionein.

Additional regulatory sequences can also be included in preferredvectors. Preferred examples of suitable regulatory sequences arerepresented by the Shine-Dalgarno of the replicase gene of the phageMS-2 and of the gene cII of bacteriophage lambda. The Shine-Dalgarnosequence may be directly followed by DNA encoding nGPCR-x and result inthe expression of the mature nGPCR-x protein.

Moreover, suitable expression vectors can include an appropriate markerthat allows the screening of the transformed host cells. Thetransformation of the selected host is carried out using any one of thevarious techniques well known to the expert in the art and described inSambrook et al., supra.

An origin of replication can also be provided either by construction ofthe vector to include an exogenous origin or may be provided by the hostcell chromosomal replication mechanism. If the vector is integrated intothe host cell chromosome, the latter may be sufficient. Alternatively,rather than using vectors which contain viral origins of replication,one skilled in the art can transform mammalian cells by the method ofco-transformation with a selectable marker and nGPCR-x DNA. An exampleof a suitable marker is dihydrofolate reductase (DHFR) or thymidinekinase (see, U.S. Pat. No. 4,399,216).

Nucleotide sequences encoding GPCR-x may be recombined with vector DNAin accordance with conventional techniques, including blunt-ended orstaggered-ended termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesiderable joining, andligation with appropriate ligases. Techniques for such manipulation aredisclosed by Sambrook et al., supra and are well known in the artMethods for construction of mammalian expression vectors are disclosedin, for example, Okayama et al, Mol. Cell. Biol., 1983, 3, 280, Cosmanet al., Mol. Immutol., 1986, 23, 935, Cosman et al., Nature, 1984, 312,768, EP-A-0367566, and WO 91/18982, each of which is incorporated hereinby reference in its entirety.

Host Cells

According to another aspect of the invention, host cells are provided,including prokaryotic and eukaryotic cells, comprising a polynucleotideof the invention (or vector of the invention) in a manner that permitsexpression of the encoded nGPCR-x polypeptide. Polynucleotides of theinvention may be introduced into the host cell as part of a circularplasmid, or as linear DNA comprising an isolated protein coding regionor a viral vector. Methods for introducing DNA into the host cell thatare well known and routinely practiced in the art includetransformation, transfection, electroporation, nuclear injection, orfusion with carriers such as liposomes, micelles, ghost cells, andprotoplasts. Expression systems of the invention include bacterial,yeast, fungal, plant, insect, invertebrate, vertebrate, and mammaliancells systems.

The invention provides host cells that are transformed or transfected(stably or transiently) with polynucleotides of the invention or vectorsof the invention. As stated above, such host cells are useful foramplifying the polynucleotides and also for expressing the nGPCR-xpolypeptide or fragment thereof encoded by the polynucleotide.

In still another related embodiment, the invention provides a method forproducing a nGPCR-x polypeptide (or fragment thereof) comprising thesteps of growing a host cell of the invention in a nutrient medium andisolating the polypeptide or variant thereof from the cell or themedium. Because nGPCR-x is a seven transmembrane receptor, it will beappreciated that, for some applications, such as certain activityassays, the preferable isolation may involve isolation of cell membranescontaining the polypeptide embedded therein, whereas for otherapplications a more complete isolation may be preferable.

According to some aspects of the present invention, transformed hostcells having an expression vector comprising any of the nucleic acidmolecules described above are provided. Expression of the nucleotidesequence occurs when the expression vector is introduced into anappropriate host cell. Suitable host cells for expression of thepolypeptides of the invention include, but are not limited to,prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vectoris employed, then the appropriate host cell would be any prokaryoticcell capable of expressing the cloned sequences. Suitable prokaryoticcells include, but are not limited to, bacteria of the generaEscherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, andStaphylococcus.

If an eukaryotic expression vector is employed, then the appropriatehost cell would be any eukaryotic cell capable of expressing the clonedsequence. Preferably, eukaryotic cells are cells of higher eukaryotes.Suitable eukaryotic cells include, but are not limited to, non-humanmammalian tissue culture cells and human tissue culture cells. Preferredhost cells include, but are not limited to, insect cells, HeLa cells,Chinese hamster ovary cells (CHO cells), African green monkey kidneycells (COS cells), human 293 cells, and murine 3T3 fibroblasts.Propagation of such cells in cell culture has become a routine procedure(see, Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973),which is incorporated herein by reference in its entirety).

In addition, a yeast host may be employed as a host cell. Preferredyeast cells include, but are not limited to, the genera Saccharomyces,Pichia, and Kluveromyces. Preferred yeast hosts are S. cerevisiae and P.pastoris. Preferred yeast vectors can contain an origin of replicationsequence from a 2T yeast plasmid, an autonomously replication sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Shuttle vectorsfor replication in both yeast and E. coli are also included herein.

Alternatively, insect cells may be used as host cells. In a preferredembodiment, the polypeptides of the invention are expressed using abaculovirus expression system (sea Luckow et al., Bio/Technology, 1988,6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O'Rielly etal (Eds.), W.H. Freeman and Company, New York, 1992, and U.S. Pat. No.4,879,236, each of which is incorporated herein by reference in itsentirety). In addition, the MAXBAC™ complete baculovirus expressionsystem (Invitrogen) can, for example, be used for production in insectcells.

Host cells of the invention are a valuable source of immunogen fordevelopment of antibodies specifically immunoreactive with nGPCR-x. Hostcells of the invention are also useful in methods for the large-scaleproduction of nGPCR-x polypeptides wherein the cells are grown in asuitable culture medium and the desired polypeptide products areisolated from the cells, or from the medium in which the cells aregrown, by purification methods known in the art, e.g., conventionalchromatographic methods including immunoaffinity chromatography,receptor affinity chromatography, hydrophobic interactionchromatography, lectin affinity chromatography, size exclusionfiltration, cation or anion exchange chromatography, high pressureliquid chromatography (HPLC), reverse phase HPLC, and the like. Stillother methods of purification include those methods wherein the desiredprotein is expressed and purified as a fusion protein having a specifictag, label, or chelating moiety that is recognized by a specific bindingpartner or agent. The purified protein can be cleaved to yield thedesired protein, or can be left as an intact fusion protein. Cleavage ofthe fusion component may produce a form of the desired protein havingadditional amino acid residues as a result of the cleavage process.

Knowledge of nGPCR-x DNA sequences allows for modification of cells topermit, or increase, expression of endogenous nGPCR-x. Cells can bemodified (e.g., by homologous recombination) to provide increasedexpression by replacing, in whole or in part, the naturally occurringnGPCR-x promoter with all or part of a heterologous promoter so that thecells express nGPCR-x at higher levels. The heterologous promoter isinserted in such a manner that it is operatively linked to endogenousnGPCR-x encoding sequences. (See, for example, PCT InternationalPublication No. WO 94/12650, PCT International Publication No. WO92/20808, and PCT International Publication No. WO 91/09955.) It is alsocontemplated that, in addition to heterologous promoter DNA, amplifiablemarker DNA (e.g., ada, dhfr, and the multifunctional CAD gene whichencodes carbamoyl phosphate synthase, aspartate transcarbamylase, anddihydroorotase) and/or intron DNA may be inserted along with theheterologous promoter DNA. If linked to the nGPCR-x coding sequence,amplification of the marker DNA by standard selection methods results inco-amplification of the nGPCR-x coding sequences in the cells.

Knockouts

The DNA sequence information provided by the present invention alsomakes possible the development (e.g., by homologous recombination or“knock-out” strategies; see Capecchi, Science 244:1288-1292 (1989),which is incorporated herein by reference) of animals that fail toexpress functional nGPCR-x or that express a variant of nGPCR-x. Suchanimals (especially small laboratory animals such as rats, rabbits, andmice) are useful as models for studying the in vivo activities ofnGPCR-x and modulators of nGPCR-x.

Antisense

Also made available by the invention are anti-sense polynucleotides thatrecognize and hybridize to polynucleotides encoding nGPCR-x. Full-lengthand fragment anti-sense polynucleotides are provided. Fragment antisensemolecules of the invention include (i) those that specifically recognizeand hybridize to nGPCR-x RNA (as determined by sequence comparison ofDNA encoding nGPCR-x to DNA encoding other known molecules).Identification of sequences unique to nGPCR-x encoding polynucleotidescan be deduced through use of any publicly available sequence database,and/or through use of commercially available sequence comparisonprograms. After identification of the desired sequences, isolationthrough restriction digestion or amplification using any of the variouspolymerase chain reaction techniques well known in the art can beperformed. Antisense polynucleotides are particularly relevant toregulating expression of nGPCR-x by those cells expressing nGPCR-x mRNA.

Antisense nucleic acids (preferably 10 to 30 base-pair oligonucleotides)capable of specifically binding to nGPCR-x expression control sequencesor nGPCR-x RNA are introduced into cells (e.g., by a viral vector orcolloidal dispersion system such as a liposome). The antisense nucleicacid binds to the nGPCR-x target nucleotide sequence in the cell andprevents transcription and/or translation of the target sequence.Phosphorothioate and methylphosphonate antisense oligonucleotides arespecifically contemplated for therapeutic use by the invention. Theantisense oligonucleotides may be further modified by addingpoly-L-lysine, transferrin polylysine, or cholesterol moieties at their5′ end. Suppression of nGPCR-x expression at either the transcriptionalor translational level is useful to generate cellular or animal modelsfor diseases/conditions characterized by aberrant nGPCR-x expression.

Antisense oligonucleotides, or fragments of odd numbered nucleotidesequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 andSEQ ID NO:191, or sequences complementary or homologous thereto, derivedfrom the nucleotide sequences of the present invention encoding nGPCR-xare useful as diagnostic tools for probing gene expression in varioustissues. For example, tissue can be probed in situ with oligonucleotideprobes carrying detectable groups by conventional autoradiographytechniques to investigate native expression of this enzyme orpathological conditions relating thereto. Antisense oligonucleotides arepreferably directed to regulatory regions of odd numbered nucleotidesequences ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 andSEQ ID NO:191, or mRNA corresponding thereto, including, but not limitedto, the initiation codon, TATA box, enhancer sequences, and the like.

Transcription Factors

The nGPCR-x sequences taught in the present invention facilitate thedesign of novel transcription factors for modulating nGPCR-x expressionin native cells and animals, and cells transformed or transfected withnGPCR-x polynucleotides. For example, the Cys₂-His₂ zinc fingerproteins, which bind DNA via their zinc finger domains, have been shownto be amenable to structural changes that lead to the recognition ofdifferent target sequences. These artificial zinc finger proteinsrecognize specific target sites with high affinity and low dissociationconstants, and are able to act as gene switches to modulate geneexpression. Knowledge of the particular nGPCR-x target sequence of thepresent invention facilitates the engineering of zinc finger proteinsspecific for the target sequence using known methods such as acombination of structure-based modeling and screening of phage displaylibraries (Segal et al., Proc. Natl. Acad. Sci. (USA) 96:2758-2763(1999); Liu et al, Proc. Natl. Acad. Sci. (USA) 94:5525-5530 (1997);Greisman et al., Science 275:657-661 (1997); Choo et al., J. Mol. Biol.273:525-532 (1997)). Each zinc finger domain usually recognizes three ormore base pairs. Since a recognition sequence of 18 base pairs isgenerally sufficient in length to render it unique in any known genome,a zinc finger protein consisting of 6 tandem repeats of zinc fingerswould be expected to ensure specificity for a particular sequence (Segalet al.) The artificial zinc finger repeats, designed based on nGPCR-xsequences, are fused to activation or repression domains to promote orsuppress nGPCR-x expression (Liu et al.) Alternatively, the zinc fingerdomains can be fused to the TATA box-binding factor (TBP) with varyinglengths of linker region between the zinc finger peptide and the TBP tocreate either transcriptional activators or repressors (Kim et al.,Proc. Natl. Acad. Sci. (USA) 94:3616-3620 (1997). Such proteins andpolynucleotides that encode them, have utility for modulating nGPCR-xexpression in vivo in both native cells, animals and humans; and/orcells transfected with nGPCR-x-encoding sequences. The noveltranscription factor can be delivered to the target cells bytransfecting constructs that express the transcription factor (genetherapy), or by introducing the protein. Engineered zinc finger proteinscan also be designed to bind RNA sequences for use in therapeutics asalternatives to antisense or catalytic RNA methods (McColl et al., Proc.Natl. Acad. Sci. (USA) 96:9521-9526 (1997); Wu et al., Proc. Natl. Acad.Sci. (USA) 92:344-348 (1995)). The present invention contemplatesmethods of designing such transcription factors based on the genesequence of the invention, as well as customized zinc finger proteins,that are useful to modulate nGPCR-x expression in cells (native ortransformed) whose genetic complement includes these sequences.

Polypeptides

The invention also provides purified and isolated mammalian nGPCR-xpolypeptides encoded by a polynucleotide of the invention. Presentlypreferred is a human nGPCR-x polypeptide comprising the amino acidsequence set out in even numbered sequences ranging from SEQ ID NO: 2 toSEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO:192, or fragments thereofcomprising an epitope specific to the polypeptide. By “epitope specificto” is meant a portion of the nGPCR receptor that is recognizable by anantibody that is specific for the nGPCR, as defined in detail below.

Although the sequences provided are particular human sequences, theinvention is intended to include within its scope other human allelicvariants; non-human mammalian forms of nGPCR-x, and other vertebrateforms of nGPCR-x.

It will be appreciated that extracellular epitopes are particularlyuseful for generating and screening for antibodies and other bindingcompounds that bind to receptors such as nGPCR-x. Thus, in anotherpreferred embodiment, the invention provides a purified and isolatedpolypeptide comprising at least one extracellular domain (e.g., theN-terminal extracellular domain or one of the three extracellular loops)of nGPCR-x. Purified and isolated polypeptides comprising the N-terminalextracellular domain of nGPCR-x are highly preferred. Also preferred isa purified and isolated polypeptide comprising a nGPCR-x fragmentselected from the group consisting of the N-terminal extracellulardomain of nGPCR-x, transmembrane domains of nGPCR-x, an extracellularloop connecting transmembrane domains of nGPCR-x, an intracellular loopconnecting transmembrane domains of nGPCR-x, the C-terminal cytoplasmicregion of nGPCR-x, and fusions thereof. Such fragments may be continuousportions of the native receptor. However, it will also be appreciatedthat knowledge of the nGPCR-x gene and protein sequences as providedherein permits recombining of various domains that are not contiguous inthe native protein. Using a FORTRAN computer program called “tmtrestall” [Parodi et al., Comput. Appl. Biosci. 5:527-535 (1994)], nGPCR-xwas shown to contain transmembrane-spanning domains.

The invention also embraces polypeptides that have at least 99%, atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55% or at least 50%identity and/or homology to the preferred polypeptide of the invention.Percent amino acid sequence “identity” with respect to the preferredpolypeptide of the invention is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical withthe residues in the nGPCR-x sequence after aligning both sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Percent sequence “homology” with respect to thepreferred polypeptide of the invention is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the residues in the nGPCR-x sequence after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and also considering any conservativesubstitutions as part of the sequence identity.

In one aspect, percent homology is calculated as the percentage of aminoacid residues in the smaller of two sequences which align with identicalamino acid residue in the sequence being compared, when four gaps in alength of 100 amino acids may be introduced to maximize alignment[Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124,National Biochemical Research Foundation, Washington, D.C. (1972),incorporated herein by reference].

Polypeptides of the invention may be isolated from natural cell sourcesor may be chemically synthesized, but are preferably produced byrecombinant procedures involving host cells of the invention. Use ofmammalian host cells is expected to provide for such post-translationalmodifications (e.g., glycosylation, truncation, lipidation, andphosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the invention. Glycosylated andnon-glycosylated forms of nGPCR-x polypeptides are embraced by theinvention.

The invention also embraces variant (or analog) nGPCR-x polypeptides. Inone example, insertion variants are provided wherein one or more aminoacid residues supplement a nGPCR-x amino acid sequence. Insertions maybe located at either or both termini of the protein, or may bepositioned within internal regions of the nGPCR-x amino acid sequence.Insertional variants with additional residues at either or both terminican include, for example, fusion proteins and proteins including aminoacid tags or labels.

Insertion variants include nGPCR-x polypeptides wherein one or moreamino acid residues are added to a nGPCR-x acid sequence or to abiologically active fragment thereof.

Variant products of the invention also include mature nGPCR-x products,ie., nGPCR-x products wherein leader or signal sequences are removed,with additional amino terminal residues. The additional amino terminalresidues may be derived from another protein, or may include one or moreresidues that are not identifiable as being derived from specificproteins. nGPCR-x products with an additional methionine residue atposition −1 (Met⁻¹-nGPCR-x) are contemplated, as are variants withadditional methionine and lysine residues at positions −2 and −1(Met⁻²-Lys⁻¹-nGPCR-x). Variants of nGPCR-x with additional Met, Met-Lys,Lys residues (or, one or more basic residues in general) areparticularly useful for enhanced recombinant protein production inbacterial host cells.

The invention also embraces nGPCR-x variants having additional aminoacid residues that result from use of specific expression systems. Forexample, use of commercially available vectors that express a desiredpolypeptide as part of a glutathione-S-transferase (GST) fusion productprovides the desired polypeptide having an additional glycine residue atposition −1 after cleavage of the GST component from the desiredpolypeptide. Variants that result from expression in other vectorsystems are also contemplated.

Insertional variants also include fusion proteins wherein the aminoterminus and/or the carboxy terminus of nGPCR-x is/are fused to anotherpolypeptide.

In another aspect, the invention provides deletion variants wherein oneor more amino acid residues in a nGPCR-x polypeptide are removed.Deletions can be effected at one or both termini of the nGPCR-xpolypeptide, or with removal of one or more non-terminal amino acidresidues of nGPCR-x. Deletion variants, therefore, include all fragmentsof a nGPCR-x polypeptide.

The invention also embraces polypeptide fragments of the even numberedsequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 andSEQ ID NO: 192, wherein the fragments maintain biological (e.g., ligandbinding and/or intracellular signaling) immunological properties of anGPCR-x polypeptide.

In one preferred embodiment of the invention, an isolated nucleic acidmolecule comprises a nucleotide sequence that encodes a polypeptidecomprising an amino acid sequence homologous to even numbered sequencesselected from the group consisting of: SEQ ID NO:2 to SEQ ID NO:94, SEQID NO: 186 and SEQ ID NO:192, and fragments thereof, wherein the nucleicacid molecule encoding at least a portion of nGPCR-x. In a morepreferred embodiment, the isolated nucleic acid molecule comprises asequence that encodes a polypeptide comprising even numbered sequencesselected from the group consisting of SEQ ID NO:2 to SEQ ID NO: 94, SEQID NO: 186 and SEQ ID NO: 192, and fragments thereof.

As used in the present invention, polypeptide fragments comprise atleast 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of theeven numbered sequences ranging from SEQ ID NO: 2 to SEQ ID NO: 94, SEQID NO: 186 and SEQ ID NO:192. Preferred polypeptide fragments displayantigenic properties unique to, or specific for, human nGPCR-x and itsallelic and species homologs. Fragments of the invention having thedesired biological and immunological properties can be prepared by anyof the methods well known and routinely practiced in the art.

In still another aspect, the invention provides substitution variants ofnGPCR-x polypeptides. Substitution variants include those polypeptideswherein one or more amino acid residues of a nGPCR-x polypeptide areremoved and replaced with alternative residues. In one aspect, thesubstitutions are conservative in nature; however, the inventionembraces substitutions that are also non-conservative. Conservativesubstitutions for this purpose may be defined as set out in Tables 2, 3,or 4 below.

Variant polypeptides include those wherein conservative substitutionshave been introduced by modification of polynucleotides encodingpolypeptides of the invention. Amino acids can be classified accordingto physical properties and contribution to secondary and tertiaryprotein structure. A conservative substitution is recognized in the artas a substitution of one amino acid for another amino acid that hassimilar properties. Exemplary conservative substitutions are set out inTable 2 (from WO 97/09433, page 10, published Mar. 13, 1997(PCT/GB96/02197, filed Sep. 6, 1996), immediately below. TABLE 2Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINO ACIDAliphatic Non-polar GAP ILV Polar - uncharged CSTM NQ Polar - charged DEKR Aromatic HFWY Other NQDE

Alternatively, conservative amino acids can be grouped as described inLehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY,N.Y. (1975), pp.71-77] as set out in Table 3, below.

Table 3 Conservative Substitutions II

TABLE 3 Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: ALIVP B. Aromatic: FW C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: STYB. Amides: NQ C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): KRH Negatively Charged (Acidic): DE

As still another alternative, exemplary conservative substitutions areset out in Table 4, below. TABLE 4 Conservative Substitutions IIIOriginal Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R)Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q)Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala,Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu,Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) SerTrp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

It should be understood that the definition of polypeptides of theinvention is intended to include polypeptides bearing modificationsother than insertion, deletion, or substitution of amino acid residues.By way of example, the modifications may be covalent in nature, andinclude for example, chemical bonding with polymers, lipids, otherorganic, and inorganic moieties. Such derivatives may be prepared toincrease circulating half-life of a polypeptide, or may be designed toimprove the targeting capacity of the polypeptide for desired cells,tissues, or organs. Similarly, the invention further embraces nGPCR-xpolypeptides that have been covalently modified to include one or morewater-soluble polymer attachments such as polyethylene glycol,polyoxyethylene glycol, or polypropylene glycol. Variants that displayligand binding properties of native nGPCR-x and are expressed at higherlevels, as well as variants that provide for constitutively activereceptors, are particularly useful in assays of the invention; thevariants are also useful in providing cellular, tissue and animal modelsof diseases/conditions characterized by aberrant nGPCR-x activity.

In a related embodiment, the present invention provides compositionscomprising purified polypeptides of the invention. Preferredcompositions comprise, in addition to the polypeptide of the invention,a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid,semisolid, or solid diluent that serves as a pharmaceutical vehicle,excipient, or medium. Any diluent known in the art may be used.Exemplary diluents include, but are not limited to, water, salinesolutions, polyoxyethylene sorbitan monolaurate, magnesium stearate,methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose,sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate,mineral oil, and cocoa butter.

Variants that display ligand binding properties of native nGPCR-x andare expressed at higher levels, as well as variants that provide forconstitutively active receptors, are particularly useful in assays ofthe invention; the variants are also useful in assays of the inventionand in providing cellular, tissue and animal models ofdiseases/conditions characterized by aberrant nGPCR-x activity.

The G protein-coupled receptor functions through a specificheterotrimeric guanine-nucleotide-binding regulatory protein (G-protein)coupled to the intracellular portion of the G protein-coupled receptormolecule. Accordingly, the G protein-coupled receptor has a specificaffinity to G protein. G proteins specifically bind to guaninenucleotides. Isolation of G proteins provides a means to isolate guaninenucleotides. G Proteins may be isolated using commercially availableanti-G protein antibodies or isolated G protein-coupled receptors.Similarly, G proteins may be detected in a sample isolated usingcommercially available detectable anti-G protein antibodies or isolatedG protein-coupled receptors.

According to the present invention, the isolated n-GPCR-x proteins ofthe present invention are useful to isolate and purify G proteins fromsamples such as cell lysates. Example 15 below sets forth an example ofisolation of G proteins using isolated nGPCR-x proteins. Such methodolgymay be used in place of the use of commercially available ant-G proteinantibodies which are used to isolate G proteins. Moreover, G proteinsmay be detected using nGPCR-x proteins in place of commerciallyavailable detectable anti-G protein antibodies. Since nGPCR-x proteinsspecifically bind to G proteins, they can be employed in any specificuse where G protein specific affinity is required, such as those useswhere commercially available anti-G protein antibodies are employed.

Antibodies

Also comprehended by the present invention are antibodies (e.g.monoclonal and polyclonal antibodies, single chain antibodies, chimericantibodies, bifunctional/bispecific antibodies, humanized antibodies,human antibodies, and complementary determining region (CDR)-graftedantibodies, including compounds which include CDR sequences whichspecifically recognize a polypeptide of the invention) specific fornGPCR-x or fragments thereof. Preferred antibodies of the invention arehuman antibodies that are produced and identified according to methodsdescribed in WO93/11236, published Jun. 20, 1993, which is incorporatedherein by reference in its entirety. Antibody fragments, including Fab,Fab′, F(ab′)₂, and F_(v), are also provided by the invention. The term“specific for,” when used to describe antibodies of the invention,indicates that the variable regions of the antibodies of the inventionrecognize and bind nGPCR-x polypeptides exclusively (i.e., are able todistinguish nGPCR-x polypeptides from other known GPCR polypeptides byvirtue of measurable differences in binding affinity, despite thepossible existence of localized sequence identity, homology, orsimilarity between nGPCR-x and such polypeptides). It will be understoodthat specific antibodies may also interact with other proteins (forexample, S. aureus protein A or other antibodies in ELISA techniques)through interactions with sequences outside the variable region of theantibodies, and, in particular, in the constant region of the molecule.Screening assays to determine binding specificity of an antibody of theinvention are well known and routinely practiced in the art. For acomprehensive discussion of such assays, see Harlow et al. (Eds.),Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; ColdSpring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize andbind fragments of the nGPCR-x polypeptides of the invention are alsocontemplated, provided that the antibodies are specific for nGPCR-xpolypeptides. Antibodies of the invention can be produced using anymethod well known and routinely practiced in the art.

The invention provides an antibody that is specific for the nGPCR-x ofthe invention. Antibody specificity is described in greater detailbelow. However, it should be emphasized that antibodies that can begenerated from polypeptides that have previously been described in theliterature and that are capable of fortuitously cross-reacting withnGPCR-x (e.g., due to the fortuitous existence of a similar epitope inboth polypeptides) are considered “cross-reactive” antibodies. Suchcross-reactive antibodies are not antibodies that are “specific” fornGPCR-x. The determination of whether an antibody is specific fornGPCR-x or is cross-reactive with another known receptor is made usingany of several assays, such as Western blotting assays, that are wellknown in the art. For identifying cells that express nGPCR-x and alsofor modulating nGPCR-x-ligand binding activity, antibodies thatspecifically bind to an extracellular epitope of the nGPCR-x arepreferred.

In one preferred variation, the invention provides monoclonalantibodies. Hybridomas that produce such antibodies also are intended asaspects of the invention. In yet another variation, the inventionprovides a humanized antibody. Humanized antibodies are useful for invivo therapeutic indications.

In another variation, the invention provides a cell-free compositioncomprising polyclonal antibodies, wherein at least one of the antibodiesis an antibody of the invention specific for nGPCR-x. Antisera isolatedfrom an animal is an exemplary composition, as is a compositioncomprising an antibody fraction of an antisera that has been resuspendedin water or in another diluent, excipient, or carrier.

In still another related embodiment, the invention provides ananti-idiotypic antibody specific for an antibody that is specific fornGPCR-x.

It is well known that antibodies contain relatively small antigenbinding domains that can be isolated chemically or by recombinanttechniques. Such domains are useful nGPCR-x binding moleculesthemselves, and also may be reintroduced into human antibodies, or fusedto toxins or other polypeptides. Thus, in still another embodiment, theinvention provides a polypeptide comprising a fragment of anGPCR-x-specific antibody, wherein the fragment and the polypeptide bindto the nGPCR-x. By way of non-limiting example, the invention providespolypeptides that are single chain antibodies and CDR-graftedantibodies.

Non-human antibodies may be humanized by any of the methods known in theart. In one method, the non-human CDRs are inserted into a humanantibody or consensus antibody framework sequence. Further changes canthen be introduced into the antibody framework to modulate affinity orimmunogenicity.

Antibodies of the invention are useful for, e.g., therapeutic purposes(by modulating activity of nGPCR-x), diagnostic purposes to detect orquantitate nGPCR-x, and purification of nGPCR-x. Kits comprising anantibody of the invention for any of the purposes described herein arealso comprehended. In general, a kit of the invention also includes acontrol antigen for which the antibody is immunospecific.

Compositions

Mutations in the nGPCR-x gene that result in loss of normal function ofthe nGPCR-x gene product underlie nGPCR-x-related human disease states.The invention comprehends gene therapy to restore nGPCR-x activity totreat those disease states. Delivery of a functional nGPCR-x gene toappropriate cells is effected ex vivo, in situ, or in vivo by use ofvectors, and more particularly viral vectors (e.g., adenovirus,adeno-associated virus, or a retrovirus), or er vivo by use of physicalDNA transfer methods (erg, liposomes or chemical treatments). See, forexample, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20(1998). For additional reviews of gene therapy technology see Friedmann,Science, 244: 1275-1281 (1989); Verna, Scientific American: 68-84(1990); and Miller, Nature, 357: 455-460 (1992). Alternatively, it iscontemplated that in other human disease states, preventing theexpression of, or inhibiting the activity of, nGPCR-x will be useful intreating disease states. It is contemplated that antisense therapy orgene therapy could be applied to negatively regulate the expression ofnGPCR-x.

Another aspect of the present invention is directed to compositions,including pharmaceutical compositions, comprising any of the nucleicacid molecules or recombinant expression vectors described above and anacceptable carrier or diluent. Preferably, the carrier or diluent ispharmaceutically acceptable. Suitable carriers are described in the mostrecent edition of Remington's Pharmaceutical Sciences, A. Osol, astandard reference text in this field, which is incorporated herein byreference in its entirety. Preferred examples of such carriers ordiluents include, but are not limited to, water, saline, Ringer'ssolution, dextrose solution, and 5% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils may also be used. Theformulations are sterilized by commonly used techniques.

Also within the scope of the invention are compositions comprisingpolypeptides, polynucleotides, or antibodies of the invention that havebeen formulated with, e.g., a pharmaceutically acceptable carrier.

The invention also provides methods of using antibodies of theinvention. For example, the invention provides a method for modulatingligand binding of a nGPCR-x comprising the step of contacting thenGPCR-x with an antibody specific for the nGPCR-x, under conditionswherein the antibody binds the receptor.

GPCRs that may be expressed in the brain, such as nGPCR-x, provide anindication that aberrant nGPCR-x signaling activity may correlate withone or more neurological or psychological disorders. The invention alsoprovides a method for treating a neurological or psychiatric disordercomprising the step of administering to a mammal in need of suchtreatment an amount of an antibody-like polypeptide of the inventionthat is sufficient to modulate ligand binding to a nGPCR-x in neurons ofthe mammal. nGPCR-x may also be expressed in other tissues, includingbut not limited to, peripheral blood lymphocytes, pancreas, ovary,uterus, testis, salivary gland, thyroid gland, kidney, adrenal gland,liver, bone marrow, prostate, fetal liver, colon, muscle, and fetalbrain, and may be found in many other tissues. Within the brain, nGPCR-xmRNA transcripts may be found in many tissues, including, but notlimited to, frontal lobe, hypothalamus, pons, cerebellum, caudatenucleus, and medulla. Tissues and brain regions where specific nGPCRs ofthe present invention are expressed are identified in the Examplesbelow.

Kits

The present invention is also directed to kits, including pharmaceuticalkits. The kits can comprise any of the nucleic acid molecules describedabove, any of the polypeptides described above, or any antibody whichbinds to a polypeptide of the invention as described above, as well as anegative control. The kit preferably comprises additional components,such as, for example, instructions, solid support, reagents helpful forquantification, and the like.

In another aspect, the invention features methods for detection of apolypeptide in a sample as a diagnostic tool for diseases or disorders,wherein the method comprises the steps of: (a) contacting the samplewith a nucleic acid probe which hybridizes under hybridization assayconditions to a nucleic acid target region of a polypeptide having thesequence of even numbered sequences ranging from SEQ ID NO: 2 to SEQ IDNO: 94, SEQ ID NO: 186 and SEQ ID NO: 192, said probe comprising thenucleic acid sequence encoding the polypeptide, fragments thereof, andthe complements of the sequences and fragments; and (b) detecting thepresence or amount of the probe:target region hybrid as an indication ofthe disease.

In preferred embodiments of the invention, the disease is selected fromthe group consisting of thyroid disorders (e.g. thyreotoxicosis,myxoedema); renal failure; inflammatory conditions (e.g., Crohn'sdisease); diseases related to cell differentiation and homeostasis;rheumatoid arthritis; autoimmune disorders; movement disorders; CNSdisorders (e.g., pain including migraine; stroke; psychotic andneurological disorders, including anxiety, schizophrenia, manicdepression, anxiety, generalized anxiety disorder, post-traumatic-stressdisorder, depression, bipolar disorder, delirium, dementia, severemental retardation; dyskinesias, such as Huntington's disease orTourette's Syndrome; attention disorders including ADD and ADHD, anddegenerative disorders such as Parkinson's, Alzheimer's; movementdisorders, including ataxias, supranuclear palsy, etc.); infections,such as viral infections caused by HIV-1 or HIV-2; metabolic andcardiovascular diseases and disorders (e.g., type 2 diabetes, obesity,anorexia, hypotension, hypertension, thrombosis, myocardial infarction,cardiomyopathies, atherosclerosis, etc.); proliferative diseases andcancers (e.g., different cancers such as breast, colon, lung, etc., andhyperproliferative disorders such as psoriasis, prostate hyperplasia,etc.); hormonal disorders (e.g., male/female hormonal replacement,polycystic ovarian syndrome, alopecia, etc); and sexual dysfunction,among others.

As described above and in Example 4 below, the genes encoding nGPCR-1(nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequenceSEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9,SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11(nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequenceSEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO:21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82),nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acidsequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequenceSEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO:86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ IDNO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90),nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ IDNO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94)and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, aminoacid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in braintissue indicating that these n-GPCR-x proteins are neuroreceptors. Kitsmay be designed to detect either expression of polynucleotides encodingthese proteins or the proteins themselves in order to identify tissue asbeing neurological. For example, oligonucleotide hybridization kits canbe provided which include a container having an oligonucleotide probespecific for the n-GPCR-x-specific DNA and optionally, containers withpositive and negative controls and/or instructions. Similarly, PCR kitscan be provided which include a container having primers specific forthe n-GPCR-x-specific sequences, DNA and optionally, containers withsize markers, positive and negative controls and/or instructions.

Hybridization conditions should be such that hybridization occurs onlywith the genes in the presence of other nucleic acid molecules. Understringent hybridization conditions only highly complementary nucleicacid sequences hybridize. Preferably, such conditions preventhybridization of nucleic acids having 1 or 2 mismatches out of 20contiguous nucleotides. Such conditions are defined supra.

The diseases for which detection of genes in a sample could bediagnostic include diseases in which nucleic acid (DNA and/or RNA) isamplified in comparison to normal cells. By “amplification” is meantincreased numbers of DNA or RNA in a cell compared with normal cells.

The diseases that could be diagnosed by detection of nucleic acid in asample preferably include central nervous system and metabolic diseases.The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The samples used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample that is compatiblewith the method utilized.

Alternatively, immunoassay kits can be provided which have containerscontainer having antibodies specific for the n-GPCR-x-protein andoptionally, containers with positive and negative controls and/orinstructions.

Kits may also be provided useful in the identification of GPCR bindingpartners such as natural ligands or modulators (agonists orantagonists). Substances useful for treatment of disorders or diseasespreferably show positive results in one or more in vitro assays for anactivity corresponding to treatment of the disease or disorder inquestion. Substances that modulate the activity of the polypeptidespreferably include, but are not limited to, antisense oligonucleotides,agonists and antagonists, and inhibitors of protein kinases.

Methods of Inducing Immune Response

Another aspect of the present invention is directed to methods ofinducing an immune response in a mammal against a polypeptide of theinvention by administering to the mammal an amount of the polypeptidesufficient to induce an immune response. The amount will be dependent onthe animal species, size of the animal, and the like but can bedetermined by those skilled in the art.

Methods of Identifying Ligands

The invention also provides assays to identify compounds that bindnGPCR-x. One such assay comprises the steps of: (a) contacting acomposition comprising a nGPCR-x with a compound suspected of bindingnGPCR-x; and (b) measuring binding between the compound and nGPCR-x. Inone variation, the composition comprises a cell expressing nGPCR-x onits surface. In another variation, isolated nGPCR-x or cell membranescomprising nGPCR-x are employed. The binding may be measured directly,e.g., by using a labeled compound, or may be measured indirectly byseveral techniques, including measuring intracellular signaling ofnGPCR-x induced by the compound (or measuring changes in the level ofnGPCR-x signaling).

Specific binding molecules, including natural ligands and syntheticcompounds, can be identified or developed using isolated or recombinantnGPCR-x products, nGPCR-x variants, or preferably, cells expressing suchproducts. Binding partners are useful for purifying nGPCR-x products anddetection or quantification of nGPCR-x products in fluid and tissuesamples using known immunological procedures. Binding molecules are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) biological activities of nGPCR-x, especially thoseactivities involved in signal transduction.

The DNA and amino acid sequence information provided by the presentinvention also makes possible identification of binding partnercompounds with which a nGPCR-x polypeptide or polynucleotide willinteract. Methods to identify binding partner compounds include solutionassays, in vitro assays wherein nGPCR-x polypeptides are immobilized,and cell-based assays. Identification of binding partner compounds ofnGPCR-x polypeptides provides candidates for therapeutic or prophylacticintervention in pathologies associated with nGPCR-x normal and aberrantbiological activity.

The invention includes several assay systems for identifying nGPCR-xbinding partners. In solution assays, methods of the invention comprisethe steps of (a) contacting a nGPCR-x polypeptide with one or morecandidate binding partner compounds and (b) identifying the compoundsthat bind to the nGPCR-x polypeptide. Identification of the compoundsthat bind the nGPCR-x polypeptide can be achieved by isolating thenGPCR-x polypeptide/binding partner complex, and separating the bindingpartner compound from the nGPCR-x polypeptide. An additional step ofcharacterizing the physical, biological, and/or biochemical propertiesof the binding partner compound is also comprehended in anotherembodiment of the invention. In one aspect, the nGPCR-xpolypeptide/binding partner complex is isolated using an antibodyimmunospecific for either the nGPCR-x polypeptide or the candidatebinding partner compound.

In still other embodiments, either the nGPCR-x polypeptide or thecandidate binding partner compound comprises a label or tag thatfacilitates its isolation, and methods of the invention to identifybinding partner compounds include a step of isolating the nGPCR-xpolypeptide/binding partner complex through interaction with the labelor tag. An exemplary tag of this type is a poly-histidine sequence,generally around six histidine residues, that permits isolation of acompound so labeled using nickel chelation. Other labels and tags, suchas the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known androutinely used in the art, are embraced by the invention.

In one variation of an in vitro assay, the invention provides a methodcomprising the steps of (a) contacting an immobilized nGPCR-xpolypeptide with a candidate binding partner compound and (b) detectingbinding of the candidate compound to the nGPCR-x polypeptide. In analternative embodiment, the candidate binding partner compound isimmobilized and binding of nGPCR-x is detected. Immobilization isaccomplished using any of the methods well known in the art, includingcovalent bonding to a support, a bead, or a chromatographic resin, aswell as non-covalent, high affinity interactions such as antibodybinding, or use of streptavidin/biotin binding wherein the immobilizedcompound includes a biotin moiety. Detection of binding can beaccomplished (i) using a radioactive label on the compound that is notimmobilized, (ii) using of a fluorescent label on the non-immobilizedcompound, (iii) using an antibody immunospecific for the non-immobilizedcompound, (iv) using a label on the non-immobilized compound thatexcites a fluorescent support to which the immobilized compound isattached, as well as other techniques well known and routinely practicedin the art.

The invention also provides cell-based assays to identify bindingpartner compounds of a nGPCR-x polypeptide. In one embodiment, theinvention provides a method comprising the steps of contacting a nGPCR-xpolypeptide expressed on the surface of a cell with a candidate bindingpartner compound and detecting binding of the candidate binding partnercompound to the nGPCR-x polypeptide. In a preferred embodiment, thedetection comprises detecting a calcium flux or other physiologicalevent in the cell caused by the binding of the molecule.

Another aspect of the present invention is directed to methods ofidentifying compounds that bind to either nGPCR-x or nucleic acidmolecules encoding nGPCR-x, comprising contacting nGPCR-x, or a nucleicacid molecule encoding the same, with a compound, and determiningwhether the compound binds nGPCR-x or a nucleic acid molecule encodingthe same. Binding can be determined by binding assays which are wellknown to the skilled artisan, including, but not limited to, gel-shiftassays, Western blots, radiolabeled competition assay, phage-basedexpression cloning, co-fractionation by chromatography,co-precipitation, cross linking, interaction trap/two-hybrid analysis,southwestern analysis, ELISA, and the like, which are described in, forexample, Current Protocols in Molecular Biology, 1999, John Wiley &Sons, NY, which is incorporated herein by reference in its entirety. Thecompounds to be screened include (which may include compounds which aresuspected to bind nGPCR-x, or a nucleic acid molecule encoding thesame), but are not limited to, extracellular, intracellular, biologic orchemical origin. The methods of the invention also embrace ligands,especially neuropeptides, that are attached to a label, such as aradiolabel (e.g., ¹²⁵I, ³⁵S, ³²P, ³³P, ³H), a fluorescence label, achemiluminescent label, an enzymic label and an immunogenic label.Modulators falling within the scope of the invention include, but arenot limited to, non-peptide molecules such as non-peptide mimetics,non-peptide allosteric effectors, and peptides. The nGPCR-x polypeptideor polynucleotide employed in such a test may either be free insolution, attached to a solid support, borne on a cell surface orlocated intracellularly or associated with a portion of a cell. Oneskilled in the art can, for example, measure the formation of complexesbetween nGPCR-x and the compound being tested. Alternatively, oneskilled in the art can examine the diminution in complex formationbetween nGPCR-x and its substrate caused by the compound being tested.

In another embodiment of the invention, high throughput screening forcompounds having suitable binding affinity to nGPCR-x is employed.Briefly, large numbers of different small peptide test compounds aresynthesized on a solid substrate. The peptide test compounds arecontacted with nGPCR-x and washed. Bound nGPCR-x is then detected bymethods well known in the art. Purified polypeptides of the inventioncan also be coated directly onto plates for use in the aforementioneddrug screening techniques. In addition, non-neutralizing antibodies canbe used to capture the protein and immobilize it on the solid support.

Generally, an expressed nGPCR-x can be used for HTS binding assays inconjunction with its defined ligand, in this case the correspondingneuropeptide that activates it. The identified peptide is labeled with asuitable radioisotope, including, but not limited to, ¹²⁵I, ³H, ³⁵S or³²P, by methods that are well known to those skilled in the art.Alternatively, the peptides may be labeled by well-known methods with asuitable fluorescent derivative (Baindur et al., Drug Dev. Res., 1994,33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160).Radioactive ligand specifically bound to the receptor in membranepreparations made from the cell line expressing the recombinant proteincan be detected in HTS assays in one of several standard ways, includingfiltration of the receptor-ligand complex to separate bound ligand fromunbound ligand (Williams, Med. Res. Rev., 1991, 11, 147-184; Sweetnam etal., J. Natural Products, 1993, 56, 441455). Alternative methods includea scintillation proximity assay (SPA) or a FlashPlate format in whichsuch separation is unnecessary (Nakayama, Cur. Opinion Drug Disc. Dev.,1998,1,85-91 Bosse et al., J. Biomolecular Screening, 1998, 3,285-292.). Binding of fluorescent ligands can be detected in variousways, including fluorescence energy transfer (FRET), directspectrophotofluorometric analysis of bound ligand, or fluorescencepolarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cur.Opinion Drug Disc. Dev, 1998, 1, 92-97).

Other assays may be used to identify specific ligands of a nGPCR-xreceptor, including assays that identify ligands of the target proteinthrough measuring direct binding of test ligands to the target protein,as well as assays that identify ligands of target proteins throughaffinity ultrafiltration with ion spray mass spectroscopy/HPLC methodsor other physical and analytical methods. Alternatively, such bindinginteractions are evaluated indirectly using the yeast two-hybrid systemdescribed in Fields et al., Nature, 340:245-246 (1989), and Fields etal., Trends in Genetics, 10:286-292 (1994), both of which areincorporated herein by reference. The two-hybrid system is a geneticassay for detecting interactions between two proteins or polypeptides.It can be used to identify proteins that bind to a known protein ofinterest, or to delineate domains or residues critical for aninteraction. Variations on this methodology have been developed to clonegenes that encode DNA binding proteins, to identify peptides that bindto a protein, and to screen for drugs. The two-hybrid system exploitsthe ability of a pair of interacting proteins to bring a transcriptionactivation domain into close proximity with a DNA binding domain thatbinds to an upstream activation sequence (UAS) of a reporter gene, andis generally performed in yeast. The assay requires the construction oftwo hybrid genes encoding (1) a DNA-binding domain that is fused to afirst protein and (2) an activation domain fused to a second protein.The DNA-binding domain targets the first hybrid protein to the UAS ofthe reporter gene; however, because most proteins lack an activationdomain, this DNA-binding hybrid protein does not activate transcriptionof the reporter gene. The second hybrid protein, which contains theactivation domain, cannot by itself activate expression of the reportergene because it does not bind the UAS. However, when both hybridproteins are present, the noncovalent interaction of the first andsecond proteins tethers the activation domain to the UAS, activatingtranscription of the reporter gene. For example, when the first proteinis a GPCR gene product, or fragment thereof, that is known to interactwith another protein or nucleic acid, this assay can be used to detectagents that interfere with the binding interaction. Expression of thereporter gene is monitored as different test agents are added to thesystem. The presence of an inhibitory agent results in lack of areporter signal.

The function of nGPCR-x gene products is unclear and no ligands have yetbeen found which bind the gene product. The yeast two-hybrid assay canalso be used to identify proteins that bind to the gene product. In anassay to identify proteins that bind to a nGPCR-x receptor, or fragmentthereof, a fusion polynucleotide encoding both a nGPCR-x receptor (orfragment) and a UAS binding domain (i.e., a first protein) may be used.In addition, a large number of hybrid genes each encoding a differentsecond protein fused to an activation domain are produced and screenedin the assay. Typically, the second protein is encoded by one or moremembers of a total cDNA or genomic DNA fusion library, with each secondprotein-coding region being fused to the activation domain. This systemis applicable to a wide variety of proteins, and it is not evennecessary to know the identity or function of the second bindingprotein. The system is highly sensitive and can detect interactions notrevealed by other methods; even transient interactions may triggertranscription to produce a stable mRNA that can be repeatedly translatedto yield the reporter protein.

Other assays may be used to search for agents that bind to the targetprotein. One such screening method to identify direct binding of testligands to a target protein is described in U.S. Pat. No. 5,585,277,incorporated herein by reference. This method relies on the principlethat proteins generally exist as a mixture of folded and unfoldedstates, and continually alternate between the two states. When a testligand binds to the folded form of a target protein (i.e., when the testligand is a ligand of the target protein), the target protein moleculebound by the ligand remains in its folded state. Thus, the folded targetprotein is present to a greater extent in the presence of a test ligandwhich binds the target protein, than in the absence of a ligand. Bindingof the ligand to the target protein can be determined by any method thatdistinguishes between the folded and unfolded states of the targetprotein. The function of the target protein need not be known in orderfor this assay to be performed. Virtually any agent can be assessed bythis method as a test ligand, including, but not limited to, metals,polypeptides, proteins, lipids, polysaccharides, polynucleotides andsmall organic molecules.

Another method for identifying ligands of a target protein is describedin Wieboldt et al., Anal. Chem., 69:1683-1691 (1997), incorporatedherein by reference. This technique screens combinatorial libraries of20-30 agents at a time in solution phase for binding to the targetprotein. Agents that bind to the target protein are separated from otherlibrary components by simple membrane washing. The specifically selectedmolecules that are retained on the filter are subsequently liberatedfrom the target protein and analyzed by HPLC and pneumatically assistedelectrospray (ion spray) ionization mass spectroscopy. This procedureselects library components with the greatest affinity for the targetprotein, and is particularly useful for small molecule libraries.

Other embodiments of the invention comprise using competitive screeningassays in which neutralizing antibodies capable of binding a polypeptideof the invention specifically compete with a test compound for bindingto the polypeptide. In this manner, the antibodies can be used to detectthe presence of any peptide that shares one or more antigenicdeterminants with nGPCR-x. Radiolabeled competitive binding studies aredescribed in A. H. Lin et al. Antimicrobial Agents and Chemotherapy,1997, vol. 41, no. 10. pp. 2127-2131, the disclosure of which isincorporated herein by reference in its entirety.

As described above and in Example 4 below, the genes encoding nGPCR-1(nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequenceSEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9,SEQ ID NO:77, amino acid sequence SEQ ID NO:10, SEQ ID NO:78), nGPCR-11(nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequenceSEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO:21, SEQ ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82),nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acidsequence SEQ ID NO:54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequenceSEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO:86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ IDNO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90),nGPCR-58 (nucleic acid sequence SEQ ID NO:67, SEQ ID NO:91, SEQ IDNO:93, amino acid sequence SEQ ID NO:68, SEQ ID NO: 92, SEQ ID NO:94),and nGPCR-3 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO:185, aminoacid sequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in braintissue indicating that these nGPCR-x proteins are neuroreceptors.Accordingly, natural binding partners of these molecules includeneurotransmitters.

Identification of Modulating Agents

The invention also provides methods for identifying a modulator ofbinding between a nGPCR-x and a nGPCR-x binding partner, comprising thesteps of: (a) contacting a nGPCR-x binding partner and a compositioncomprising a nGPCR-x in the presence and in the absence of a putativemodulator compound; (b) detecting binding between the binding partnerand the nGPCR-x; and (c) identifying a putative modulator compound or amodulator compound in view of decreased or increased binding between thebinding partner and the nGPCR-x in the presence of the putativemodulator, as compared to binding in the absence of the putativemodulator.

nGPCR-x binding partners that stimulate nGPCR-x activity are useful asagonists in disease states or conditions characterized by insufficientnGPCR-x signaling (e.g., as a result of insufficient activity of anGPCR-x ligand). nGPCR-x binding partners that block ligand-mediatednGPCR-x signaling are useful as nGPCR-x antagonists to treat diseasestates or conditions characterized by excessive nGPCR-x signaling. Inaddition nGPCR-x modulators in general, as well as nGPCR-xpolynucleotides and polypeptides, are useful in diagnostic assays forsuch diseases or conditions.

In another aspect, the invention provides methods for treating a diseaseor abnormal condition by administering to a patient in need of suchtreatment a substance that modulates the activity or expression of apolypeptide having the sequence of even numbered sequences ranging fromSEQ ID NO: 2 to SEQ ID NO: 94, SEQ ID NO: 186 and SEQ ID NO: 192.

Agents that modulate (i.e., increase, decrease, or block) nGPCR-xactivity or expression may be identified by incubating a putativemodulator with a cell containing a nGPCR-x polypeptide or polynucleotideand determining the effect of the putative modulator on nGPCR-x activityor expression. The selectivity of a compound that modulates the activityof nGPCR-x can be evaluated by comparing its effects on nGPCR-x to itseffect on other GPCR compounds. Selective modulators may include, forexample, antibodies and other proteins, peptides, or organic moleculesthat specifically bind to a nGPCR-x polypeptide or a nGPCR-x-encodingnucleic acid. Modulators of nGPCR-x activity will be therapeuticallyuseful in treatment of diseases and physiological conditions in whichnormal or aberrant nGPCR-x activity is involved. nGPCR-xpolynucleotides, polypeptides, and modulators may be used in thetreatment of such diseases and conditions as infections, such as viralinfections caused by HIV-1 or HIV-2; pain; cancers; Parkinson's disease;hypotension; hypertension; and psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, delirium, dementia,severe mental retardation and dyskinesias, such as Huntington's diseaseor Tourette's Syndrome, among others. nGPCR-x polynucleotides andpolypeptides, as well as nGPCR-x modulators, may also be used indiagnostic assays for such diseases or conditions.

Methods of the invention to identify modulators include variations onany of the methods described above to identify binding partnercompounds, the variations including techniques wherein a binding partnercompound has been identified and the binding assay is carried out in thepresence and absence of a candidate modulator. A modulator is identifiedin those instances where binding between the nGPCR-x polypeptide and thebinding partner compound changes in the presence of the candidatemodulator compared to binding in the absence of the candidate modulatorcompound. A modulator that increases binding between the nGPCR-xpolypeptide and the binding partner compound is described as an enhanceror activator, and a modulator that decreases binding between the nGPCR-xpolypeptide and the binding partner compound is described as aninhibitor.

The invention also comprehends high-throughput screening (HTS) assays toidentify compounds that interact with or inhibit biological activity(i.e., affect enzymatic activity, binding activity, etc.) of a nGPCR-xpolypeptide. HTS assays permit screening of large numbers of compoundsin an efficient manner. Cell-based HTS systems are contemplated toinvestigate nGPCR-x receptor-ligand interaction. HTS assays are designedto identify “hits” or “lead compounds” having the desired property, fromwhich modifications can be designed to improve the desired property.Chemical modification of the “hit” or “lead compound” is often based onan identifiable structure/activity relationship between the “hit” andthe nGPCR-x polypeptide.

Another aspect of the present invention is directed to methods ofidentifying compounds which modulate (i.e., increase or decrease)activity of nGPCR-x comprising contacting nGPCR-x with a compound, anddetermining whether the compound modifies activity of nGPCR-x. Theactivity in the presence of the test compared is measured to theactivity in the absence of the test compound. Where the activity of thesample containing the test compound is higher than the activity in thesample lacking the test compound, the compound will have increasedactivity. Similarly, where the activity of the sample containing thetest compound is lower than the activity in the sample lacking the testcompound, the compound will have inhibited activity.

The present invention is particularly useful for screening compounds byusing nGPCR-x in any of a variety of drug screening techniques. Thecompounds to be screened include (which may include compounds which aresuspected to modulate nGPCR-x activity), but are not limited to,extracellular, intracellular, biologic or chemical origin. The nGPCR-xpolypeptide employed in such a test may be in any form, preferably, freein solution, attached to a solid support, borne on a cell surface orlocated intracellularly. One skilled in the art can, for example,measure the formation of complexes between nGPCR-x and the compoundbeing tested. Alternatively, one skilled in the art can examine thediminution in complex formation between nGPCR-x and its substrate causedby the compound being tested.

The activity of nGPCR-x polypeptides of the invention can be determinedby, for example, examining the ability to bind or be activated bychemically synthesized peptide ligands. Alternatively, the activity ofnGPCR-x polypeptides can be assayed by examining their ability to bindcalcium ions, hormones, chemokines, neuropeptides, neurotransmitters,nucleotides, lipids, odorants, and photons. Alternatively, the activityof the nGPCR-x polypeptides can be determined by examining the activityof effector molecules including, but not limited to, adenylate cyclase,phospholipases and ion channels. Thus, modulators of nGPCR-x polypeptideactivity may alter a GPCR receptor function, such as a binding propertyof a receptor or an activity such as G protein-mediated signaltransduction or membrane localization. In various embodiments of themethod, the assay may take the form of an ion flux assay, a yeast growthassay, a non-hydrolyzable GTP assay such as a [³⁵S]-GTP S assay, a cAMPassay, an inositol triphosphate assay, a diacylglycerol assay, anAequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca²⁺concentration, a mitogenesis assay, a MAP Kinase activity assay, anarachidonic acid release assay (e.g., using [³H]-arachidonic acid), andan assay for extracellular acidification rates, as well as other bindingor function-based assays of nGPCR-x activity that are generally known inthe art. In several of these embodiments, the invention comprehends theinclusion of any of the G proteins known in the art, such as G₁₆, G₁₅,or chimeric G_(qd5), G_(qs5), G_(qo5), G_(q25), and the like. nGPCR-xactivity can be determined by methodologies that are used to assay forFaRP activity, which is well known to those skilled in the art.Biological activities of nGPCR-x receptors according to the inventioninclude, but are not limited to, the binding of a natural or anunnatural ligand, as well as any one of the functional activities ofGPCRs known in the art. Non-limiting examples of GPCR activities includetransmembrane signaling of various forms, which may involve G proteinassociation and/or the exertion of an influence over G protein bindingof various guanidylate nucleotides; another exemplary activity of GPCRsis the binding of accessory proteins or polypeptides that differ fromknown G proteins.

The modulators of the invention exhibit a variety of chemicalstructures, which can be generally grouped into non-peptide mimetics ofnatural GPCR receptor ligands, peptide and non-peptide allostericeffectors of GPCR receptors, and peptides that may function asactivators or inhibitors (competitive, uncompetitive andnon-competitive) (e.g., antibody products) of GPCR receptors. Theinvention does not restrict the sources for suitable modulators, whichmay be obtained from natural sources such as plant, animal or mineralextracts, or non-natural sources such as small molecule libraries,including the products of combinatorial chemical approaches to libraryconstruction, and peptide libraries. Examples of peptide modulators ofGPCR receptors exhibit the following primary structures: GLGPRPLRFamide,GNSFLRFamide, GGPQGPLRFamide, GPSGPLRFamide, PDVDHVFLRFamide, andpyro-EDVDHVFLRFamide.

Other assays can be used to examine enzymatic activity including, butnot limited to, photometric, radiometric, HPLC, electrochemical, and thelike, which are described in, for example, Enzyme Assays: A PracticalApproach, eds. R. Eisenthal and M. J. Danson, 1992, Oxford UniversityPress, which is incorporated herein by reference in its entirety.

The use of cDNAs encoding GPCRs in drug discovery programs iswell-known; assays capable of testing thousands of unknown compounds perday in high-throughput screens (HTSs) are thoroughly documented. Theliterature is replete with examples of the use of radiolabelled ligandsin HTS binding assays for drug discovery (see Williams, MedicinalResearch Reviews, 1991, 11, 147-184.; Sweetnam, et al., J. NaturalProducts, 1993, 56, 441-455 for review). Recombinant receptors arepreferred for binding assay HTS because they allow for betterspecificity (higher relative purity), provide the ability to generatelarge amounts of receptor material, and can be used in a broad varietyof formats (see Hodgson, Bio/Technology, 1992, 10, 973-980; each ofwhich is incorporated herein by reference in its entirety).

A variety of heterologous systems is available for functional expressionof recombinant receptors that are well known to those skilled in theart. Such systems include bacteria (Strosberg, et al., Trends inPharmacological Sciences, 1992, 13, 95-98), yeast (Pausch, Trends inBiotechnology, 1997, 15, 487-494), several kinds of insect cells (VandenBroeck, Int. Rev. Cytology, 1996, 164, 189-268), amphibian cells(Jayawickreme et al., Current Opinion in Biotechnology, 1997, 8, 629634)and several mammalian cell lines (CHO, BEK293, COS, etc.; see Gerhardt,et al., Eur. J. Pharmacology, 1997, 334, 1-23). These examples do notpreclude the use of other possible cell expression systems, includingcell lines obtained from nematodes (PCT application WO 98/37177).

In preferred embodiments of the invention, methods of screening forcompounds that modulate nGPCR-x activity comprise contacting testcompounds with nGPCR-x and assaying for the presence of a complexbetween the compound and nGPCR-x. In such assays, the ligand istypically labeled. After suitable incubation, free ligand is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular compound to bind tonGPCR-x.

It is well known that activation of heterologous receptors expressed inrecombinant systems results in a variety of biological responses, whichare mediated by G proteins expressed in the host cells. Occupation of aGPCR by an agonist results in exchange of bound GDP for GIT at a bindingsite on the Ga subunit; one can use a radioactive, non-hydrolyzablederivative of GTP, GTPγ[³⁵S], to measure binding of an agonist to thereceptor (Sim et al., Neuroreport, 1996, 7, 729-733). One can also usethis binding to measure the ability of antagonists to bind to thereceptor by decreasing binding of GTPγ[³⁵S] in the presence of a knownagonist. One could therefore construct a HTS based on GTPγ[³⁵S] binding,though this is not the preferred method.

The G proteins required for functional expression of heterologous GPCRscan be native constituents of the host cell or can be introduced throughwell-known recombinant technology. The G proteins can be intact orchimeric. Often, a nearly universally competent G protein (e.g.,G_(α16)) is used to couple any given receptor to a detectable responsepathway. G protein activation results in the stimulation or inhibitionof other native proteins, events that can be linked to a measurableresponse.

Examples of such biological responses include, but are not limited to,the following: the ability to survive in the absence of a limitingnutrient in specifically engineered yeast cells (Pausch, Trends inBiotechnology, 1997, 15, 487-494); changes in intracellular Ca²⁺concentration as measured by fluorescent dyes (Murphy, et al, Cur.Opinion Drug Disc. Dev., 1998, 1, 192-199). Fluorescence changes canalso be used to monitor ligand-induced changes in membrane potential orintracellular pH; an automated system suitable for HTS has beendescribed for these purposes (Schroeder, et al., J. BiomolecularScreening, 1996, 1, 75-80). Melanophores prepared from Xenopus laevisshow a ligand-dependent change in pigment organization in response toheterologous GPCR activation; this response is adaptable to HTS formats(Jayawickreme et al, Cur. Opinion Biotechnology, 1997, 8, 629634).Assays are also available for the measurement of common secondmessengers, including cAMP, phosphoinositides and arachidonic acid, butthese are not generally preferred for HTS.

Preferred methods of HTS employing these receptors include permanentlytransfected CHO cells, in which agonists and antagonists can beidentified by the ability to specifically alter the binding of GTPγ[³⁵S]in membranes prepared from these cells. In another embodiment of theinvention, permanently transfected CHO cells could be used for thepreparation of membranes which contain significant amounts of therecombinant receptor proteins; these membrane preparations would then beused in receptor binding assays, employing the radiolabelled ligandspecific for the particular receptor. Alternatively, a functional assay,such as fluorescent monitoring of ligand-induced changes in internalCa²⁺ concentration or membrane potential in permanently transfected CHOcells containing each of these receptors individually or in combinationwould be preferred for HTS. Equally preferred would be an alternativetype of mammalian cell, such as HEK293 or COS cells, in similar formats.More preferred would be permanently transfected insect cell lines, suchas Drosophila S2 cells. Even more preferred would be recombinant yeastcells expressing the Drosophila melanogaster receptors in HTS formatswell known to those skilled in the art (e.g., Pausch, Trends inBiotechnology, 1997, 15, 487-494).

The invention contemplates a multitude of assays to screen and identifyinhibitors of ligand binding to nGPCR-x receptors. In one example, thenGPCR-x receptor is immobilized and interaction with a binding partneris assessed in the presence and absence of a candidate modulator such asan inhibitor compound. In another example, interaction between thenGPCR-x receptor and its binding partner is assessed in a solutionassay, both in the presence and absence of a candidate inhibitorcompound. In either assay, an inhibitor is identified as a compound thatdecreases binding between the nGPCR-x receptor and its binding partner.Another contemplated assay involves a variation of the dihybrid assaywherein an inhibitor of protein/protein interactions is identified bydetection of a positive signal in a transformed or transfected hostcell, as described in PCT publication number WO 95/20652, published Aug.3, 1995.

Candidate modulators contemplated by the invention include compoundsselected from libraries of either potential activators or potentialinhibitors. There are a number of different libraries used for theidentification of small molecule modulators, including: (1) chemicallibraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides or organicmolecules. Chemical libraries consist of random chemical structures,some of which are analogs of known compounds or analogs of compoundsthat have been identified as “hits” or “leads” in other drug discoveryscreens, some of which are derived from natural products, and some ofwhich arise from non-directed synthetic organic chemistry. Naturalproduct libraries are collections of microorganisms, animals, plants, ormarine organisms which are used to create mixtures for screening by: (1)fermentation and extraction of broths from soil, plant or marinemicroorganisms or (2) extraction of plants or marine organisms. Naturalproduct libraries include polyketides, non-ribosomal peptides, andvariants (non-naturally occurring) thereof. For a review, see Science282:63-68 (1998). Combinatorial libraries are composed of large numbersof peptides, oligonucleotides, or organic compounds as a mixture. Theselibraries are relatively easy to prepare by traditional automatedsynthesis methods, PCR, cloning, or proprietary synthetic methods. Ofparticular interest are non-peptide combinatorial libraries. Still otherlibraries of interest include peptide, protein, peptidomimetic,multiparallel synthetic collection, recombinatorial, and polypeptidelibraries. For a review of combinatorial chemistry and libraries createdtherefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).Identification of modulators through use of the various librariesdescribed herein permits modification of the candidate “hit” (or “lead”)to optimize the capacity of the “hit” to modulate activity.

Still other candidate inhibitors contemplated by the invention can bedesigned and include soluble forms of binding partners, as well as suchbinding partners as chimeric, or fusion, proteins. A “binding partner”as used herein broadly encompasses non-peptide modulators, as well assuch peptide modulators as neuropeptides other than natural ligands,antibodies, antibody fragments, and modified compounds comprisingantibody domains that are immunospecific for the expression product ofthe identified nGPCR-x gene.

The polypeptides of the invention are employed as a research tool foridentification, characterization and purification of interacting,regulatory proteins. Appropriate labels are incorporated into thepolypeptides of the invention by various methods known in the art andthe polypeptides are used to capture interacting molecules. For example,molecules are incubated with the labeled polypeptides, washed to removeunbound polypeptides, and the polypeptide complex is quantified. Dataobtained using different concentrations of polypeptide are used tocalculate values for the number, affinity, and association ofpolypeptide with the protein complex.

Labeled polypeptides are also useful as reagents for the purification ofmolecules with which the polypeptide interacts including, but notlimited to, inhibitors. In one embodiment of affinity purification, apolypeptide is covalently coupled to a chromatography column. Cells andtheir membranes are extracted, and various cellular subcomponents arepassed over the column. Molecules bind to the column by virtue of theiraffinity to the polypeptide. The polypeptide-complex is recovered fromthe column, dissociated and the recovered molecule is subjected toprotein sequencing. This amino acid sequence is then used to identifythe captured molecule or to design degenerate oligonucleotides forcloning the corresponding gene from an appropriate cDNA library.

Alternatively, compounds may be identified which exhibit similarproperties to the ligand for the nGPCR-x of the invention, but which aresmaller and exhibit a longer half time than the endogenous ligand in ahuman or animal body. When an organic compound is designed, a moleculeaccording to the invention is used as a “lead” compound. The design ofmimetics to known pharmaceutically active compounds is a well-knownapproach in the development of pharmaceuticals based on such “lead”compounds. Mimetic design, synthesis and testing are generally used toavoid randomly screening a large number of molecules for a targetproperty. Furthermore, structural data deriving from the analysis of thededuced amino acid sequences encoded by the DNAs of the presentinvention are useful to design new drugs, more specific and thereforewith a higher pharmacological potency.

Comparison of the protein sequence of the present invention with thesequences present in all the available databases showed a significanthomology with the transmembrane portion of G protein coupled receptors.Accordingly, computer modeling can be used to develop a putativetertiary structure of the proteins of the invention based on theavailable information of the transmembrane domain of other proteins.Thus, novel ligands based on the predicted structure of nGPCR-x can bedesigned.

In a particular embodiment, the novel molecules identified by thescreening methods according to the invention are low molecular weightorganic molecules, in which case a composition or pharmaceuticalcomposition can be prepared thereof for oral intake, such as in tablets.The compositions, or pharmaceutical compositions, comprising the nucleicacid molecules, vectors, polypeptides, antibodies and compoundsidentified by the screening methods described herein, can be preparedfor any route of administration including, but not limited to, oral,intravenous, cutaneous, subcutaneous, nasal, intramuscular orintraperitoneal. The nature of the carrier or other ingredients willdepend on the specific route of administration and particular embodimentof the invention to be administered. Examples of techniques andprotocols that are useful in this context are, inter alia, found inRemington's Pharmaceutical Sciences, 16^(th) edition, Osol, A (ed.),1980, which is incorporated herein by reference in its entirety.

The dosage of these low molecular weight compounds will depend on thedisease state or condition to be treated and other clinical factors suchas weight and condition of the human or animal and the route ofadministration of the compound. For treating human or animals, betweenapproximately 0.5 mg/kg of body weight to 500 mg/kg of body weight ofthe compound can be administered. Therapy is typically administered atlower dosages and is continued until the desired therapeutic outcome isobserved.

The present compounds and methods, including nucleic acid molecules,polypeptides, antibodies, compounds identified by the screening methodsdescribed herein, have a variety of pharmaceutical applications and maybe used, for example, to treat or prevent unregulated cellular growth,such as cancer cell and tumor growth. In a particular embodiment, thepresent molecules are used in gene therapy. For a review of gene therapyprocedures, see e.g. Anderson, Science, 1992, 256, 808-813, which isincorporated herein by reference in its entirety.

The present invention also encompasses a method of agonizing(stimulating) or antagonizing a nGPCR-x natural binding partnerassociated activity in a mammal comprising administering to said mammalan agonist or antagonist to one of the above disclosed polypeptides inan amount sufficient to effect said agonism or antagonism. Oneembodiment of the present invention, then, is a method of treatingdiseases in a mammal with an agonist or antagonist of the protein of thepresent invention comprises administering the agonist or antagonist to amammal in an amount sufficient to agonize or antagonizenGPCR-x-associated functions.

In an effort to discover novel treatments for diseases, biomedicalresearchers and chemists have designed, synthesized, and testedmolecules that inhibit the function of protein polypeptides. Some smallorganic molecules form a class of compounds that modulate the functionof protein polypeptides. Examples of molecules that have been reportedto inhibit the function of protein kinases include, but are not limitedto, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO92/20642, published Nov. 26, 1992 by Maguire et al.), vinylene-azaindolederivatives (PCT WO 94/14808, published Jul. 7, 1994 by Ballinari etal.), 1-cyclopropyl-4-pyridyluinolones (U.S. Pat. No. 5,330,992), styrylcompounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridylcompounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EPApplication No. 0 566 266 A1), seleoindoles and selenides (PCT WO94/03427, published Feb. 17, 1994 by Denny et al.), tricyclicpolyhydroxylic compounds (PCT WO 92/21660, published Dec. 10, 1992 byDow), and benzylphosphonic acid compounds (PCT WO 91/15495, publishedOct. 17, 1991 by Dow et al), all of which are incorporated by referenceherein, including any drawings.

Exemplary diseases and conditions amenable to treatment based on thepresent invention include, but are not limited to, thyroid disorders(e.g. thyreotoxicosis, myxoedema); renal failure; inflammatoryconditions (e g., Chron's disease); diseases related to celldifferentiation and homeostasis; rheumatoid arthritis; autoimmunedisorders; movement disorders; CNS disorders (e.g., pain includingmigraine; stroke; psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, anxiety, generalized anxietydisorder, post-traumatic-stress disorder, depression, bipolar disorder,delirium, dementia, severe mental retardation; dyskinesias, such asHuntington's disease or Tourette's Syndrome; attention disordersincluding ADD and ADHD, and degenerative disorders such as Parkinson's,Alzheimer's; movement disorders, including ataxias, supranuclear palsy,etc); infections, such as viral infections caused by HIV-1 or HIV-2;metabolic and cardiovascular diseases and disorders (e.g., type 2diabetes, obesity, anorexia, hypotension, hypertension, thrombosis,myocardial infarction, cardiomyopathies, atherosclerosis, etc.);proliferative diseases and cancers (e.g., different cancers such asbreast, colon, lung, etc., and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc); hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.); sexual dysfunction, among others.

Compounds that can traverse cell membranes and are resistant to acidhydrolysis are potentially advantageous as therapeutics as they canbecome highly bioavailable after being administered orally to patients.However, many of these protein inhibitors only weakly inhibit function.In addition, many inhibit a variety of protein kinases and willtherefore cause multiple side effects as therapeutics for diseases.

Some indolinone compounds, however, form classes of acid resistant andmembrane permeable organic molecules. WO 96/22976 (published Aug. 1,1996 by Ballinari et at) describes hydrosoluble indolinone compoundsthat harbor tetralin, naphthalene, quinoline, and indole substituentsfused to the oxindole ring. These bicyclic substituents are in turnsubstituted with polar groups including hydroxylated alkyl, phosphate,and ether substituents. U.S. patent application Ser. No. 08/702,232,filed Aug. 23, 1996, entitled “Indolinone Combinatorial Libraries andRelated Products and Methods for the Treatment of Disease” by Tang etal. (Lyon & Lyon Docket No. 221/187) and 08/485,323, filed Jun. 7, 1995,entitled “Benzylidene-Z-Indoline Compounds for the Treatment of Disease”by Tang et al. (Lyon & Lyon Docket No. 223/298) and International PatentPublication WO 96/22976, published Aug. 1, 1996 by Ballinari et al., allof which are incorporated herein by reference in their entirety,including any drawings, describe indolinone chemical libraries ofindolinone compounds harboring other bicyclic moieties as well asmonocyclic moieties fused to the oxindole ring. Application Ser. Nos.08/02,232, filed Aug. 23, 1996, entitled “Indolinone CombinatorialLibraries and Related Products and Methods for the Treatment of Disease”by Tang et al. (Lyon & Lyon Docket No. 221/187), 08/485,323, filed Jun.7, 1995, entitled “Benzylidene-Z-Indoline Compounds for the Treatment ofDisease” by Tang et al. (Lyon & Lyon Docket No. 223/298), and WO96/22976, published Aug. 1, 1996 by Ballinari et al. teach methods ofindolinone synthesis, methods of testing the biological activity ofindolinone compounds in cells, and inhibition patterns of indolinonederivatives, both of which are incorporated by reference herein,including any drawings.

Other examples of substances capable of modulating kinase activityinclude, but are not limited to, tyrphostins, quinazolines,quinoxolines, and quinolines. The quinazolines, tyrphostins, quinolines,and quinoxolines referred to above include well-known compounds such asthose described in the literature. For example, representativepublications describing quinazolines include Barker et al., EPOPublication No. 0 520 722 A1; Jones et al., U.S. Pat. No. 4,447,608;Kabbe et al., U.S. Pat. No. 4,757,072; Kaul and Vougioukas, U.S. Pat.No. 5,316,553; Kreighbaum and Corner, U.S. Pat. No. 4,343,940; Pegg andWardleworth, EPO Publication No. 0 562 734 A1; Barker et al., Proc. ofAm. Assoc. for Cancer Research 32:327 (1991); Bertino, J. R., CancerResearch 3:293-304 (1979); Bertino, J. R., Cancer Research 9(2 part1):293-304 (1979); Curtinet al., Br. J. Cancer 53:361-368 (1986);Fernandes et al., Cancer Research 43:1117-1123 (1983); Ferris et al. J.Org. Chem. 44(2):173-178; Fry et al., Science 265:1093-1095 (1994);Jackman et al., Cancer Research 51:5579-5586 (1981); Jones et al. J.Med. Chem. 29(6):1114-1118; Lee and Skibo, Biochemistry 26(23):7355-7362(1987); Lemus et al., J. Org. Chem. 54:3511-3518 (1989); Ley and Seng,Synthesis 1975:415-522 (1975); Maxwell et al., Magnetic Resonance inMedicine 17:189-196 (1991); Mini et al., Cancer Research 45:325-330(1985); Phillips and Castle, J. Heterocyclic Chem. 17(19):1489-1596(1980); Reece et al., Cancer Research 47(11):2996-2999 (1977); Sculieret al., Cancer Immunol. and Immunother. 23:A65 (1986); Sikora et al.,Cancer Letters 23:289-295 (1984); and Sikora et al., Analytical Biochem.172:344-355 (1988), all of which are incorporated herein by reference intheir entirety, including any drawings.

Quinoxaline is described in Kaul and Vougioukas, U.S. Pat. No.5,316,553, incorporated herein by reference in its entirety, includingany drawings.

Quinolines are described in Dolle et al., J. Med. Chem. 37:2627-2629(1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994); Burke et al., J.Med. Chem. 36:425-432 (1993); and Burke et al. BioOrganic Med. Chem.Letters 2:1771-1774 (1992), all of which are incorporated by referencein their entirety, including any drawings.

Tyrphostins are described in Allen et al., Clin. Exp. Immunol.91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker etal., J. Cell Sci. 102:543-555 (1992); Bilder et al., Amer. Physiol. Soc.pp. 6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer.Assoc. Cancer Rsch. 33:558 (1992); Bryckaert et al., Experimental CellResearch 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60(1993); Dong et al., J. Immunol. 151(5):2717-2724 (1993); Gazit et al.,J. Med. Chem. 32:2344-2352 (1989); Gazit et al., J. Med. Chem.36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994);King et al., Biochem. J. 275:413418 (1991); Kuo et al., Cancer Letters74:197-202 (1993); Levitzkd, A., The FASEB J. 6:3275-3282 (1992); Lyallet al., J. Biol. Chem. 264:14503-14509 (1989); Peterson et al., TheProstate 22:335-345 (1993); Pillemer et al., Int. J. Cancer 50:80-85(1992); Posner et al., Molecular Pharmacology 45:673-683 (1993); Renduet al., Biol. Pharmacology 44(5):881-888 (1992); Sauro and Thomas, LifeSciences 53:371-376 (1993); Sauro and Thomas, J. Pharm. and ExperimentalTherapeutics 267(3):119-1125 (1993); Wolbring et al., J. Biol. Chem.269(36):22470-22472 (1994); and Yoneda et al., Cancer Research51:44304435 (1991); all of which are incorporated herein by reference intheir entirety, including any drawings.

Other compounds that could be used as modulators include oxindolinonessuch as those described in U.S. patent application Ser. No. 08/702,232filed Aug. 23, 1996, incorporated herein by reference in its entirety,including any drawings.

Methods of determining the dosages of compounds to be administered to apatient and modes of administering compounds to an organism aredisclosed in U.S. application Ser. No. 08/702,282, filed Aug. 23, 1996and International patent publication number WO 96/22976, published Aug.1, 1996, both of which are incorporated herein by reference in theirentirety, including any drawings, figures or tables. Those skilled inthe art will appreciate that such descriptions are applicable to thepresent invention and can be easily adapted to it.

The proper dosage depends on various factors such as the type of diseasebeing treated, the particular composition being used and the size andphysiological condition of the patient. Therapeutically effective dosesfor the compounds described herein can be estimated initially from cellculture and animal models. For example, a dose can be formulated inanimal models to achieve a circulating concentration range thatinitially takes into account the IC₅₀ as determined in cell cultureassays. The animal model data can be used to more accurately determineuseful doses in humans.

Plasma half-life and biodistribution of the drug and metabolites in theplasma, tumors and major organs can also be determined to facilitate theselection of drugs most appropriate to inhibit a disorder. Suchmeasurements can be carried out. For example, HPLC analysis can beperformed on the plasma of animals treated with the drug and thelocation of radiolabeled compounds can be determined using detectionmethods such as X-ray, CAT scan and MRI. Compounds that show potentinhibitory activity in the screening assays, but have poorpharmacokinetic characteristics, can be optimized by altering thechemical structure and retesting. In this regard, compounds displayinggood pharmacokinetic characteristics can be used as a model.

Toxicity studies can also be carried out by measuring the blood cellcomposition. For example, toxicity studies can be carried out in asuitable animal model as follows: 1) the compound is administered tomice (an untreated control mouse should also be used); 2) blood samplesare periodically obtained via the tail vein from one mouse in eachtreatment group; and 3) the samples are analyzed for red and white bloodcell counts, blood cell composition and the percent of lymphocytesversus polymorphonuclear cells. A comparison of results for each dosingregime with the controls indicates if toxicity is present.

At the termination of each toxicity study, further studies can becarried out by sacrificing the animals (preferably, in accordance withthe American Veterinary Medical Association guidelines Report of theAmerican Veterinary Medical Assoc. Panel on Euthanasia, Journal ofAmerican Veterinary Medical Assoc., 202:229-249, 1993). Representativeanimals from each treatment group can then be examined by gross necropsyfor immediate evidence of metastasis, unusual illness or toxicity. Grossabnormalities in tissue are noted and tissues are examinedhistologically. Compounds causing a reduction in body weight or bloodcomponents are less preferred, as are compounds having an adverse effecton major organs. In general, the greater the adverse effect the lesspreferred the compound.

For the treatment of cancers the expected daily dose of a hydrophobicpharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250mg/day, and most preferably 1 to 50 mg/day. Drugs can be delivered lessfrequently provided plasma levels of the active moiety are sufficient tomaintain therapeutic effectiveness. Plasma levels should reflect thepotency of the drug. Generally, the more potent the compound the lowerthe plasma levels necessary to achieve efficacy.

nGPCR-x mRNA transcripts may found in many tissues, including, but notlimited to, brain, peripheral blood lymphocytes, pancreas, ovary,uterus, testis, salivary gland, kidney, adrenal gland, liver, bonemarrow, prostate, fetal liver, colon, muscle, and fetal brain, and maybe found in many other tissues. Within the brain, nGPCR-x mRNAtranscripts may be found in many tissues, including, but not limited to,frontal lobe, hypothalamus, pons, cerebellum, caudate nucleus, andmedulla. Tissues and brain regions where specific nGPCR mRNA transcriptsare expressed are identified in the Examples, below.

Odd numbered nucleotide sequences ranging from SEQ ID NO: 1 to SEQ IDNO: 93, SEQ ID NO: 185 and SEQ ID NO:191 will, as detailed above, enablescreening the endogenous neurotransmitters/hormones/ligands whichactivate, agonize, or antagonize nGPCR-x and for compounds withpotential utility in treating disorders including, but not limited to,thyroid disorders (e.g. thyreotoxicosis, myxoedema); renal failure;inflammatory conditions (e.g., Chron's disease); diseases related tocell differentiation and homeostasis; rheumatoid arthritis; autoimmunedisorders; movement disorders; CNS disorders (e.g., pain includingmigraine; stroke; psychotic and neurological disorders, includinganxiety, schizophrenia, manic depression, anxiety, generalized anxietydisorder, post-traumatic-stress disorder, depression, bipolar disorder,delirium, dementia, severe mental retardation; dyskinesias, such asHuntington's disease or Tourette's Syndrome; attention disordersincluding ADD and ADHD, and degenerative disorders such as Parkinson's,Alzheimer's; movement disorders, including ataxias, supranuclear palsy,etc.); infections, such as viral infections caused by HIV-1 or HIV-2;metabolic and cardiovascular diseases and disorders (e.g., type 2diabetes, obesity, anorexia, hypotension, hypertension, thrombosis,myocardial infarction, cardiomyopathies, atherosclerosis, etc.);proliferative diseases and cancers (e.g., different cancers such asbreast, colon, lung, etc, and hyperproliferative disorders such aspsoriasis, prostate hyperplasia, etc.); hormonal disorders (e.g.,male/female hormonal replacement, polycystic ovarian syndrome, alopecia,etc.); sexual dysfunction, among others.

For example, nGPCR-x may be useful in the treatment of respiratoryailments such as asthma, where T cells are implicated by the disease.Contraction of airway smooth muscle is stimulated by thrombin. Cicala etal (1999) Br J Pharmacol 126:478484. Additionally, in bronchiolitisobliterans, it has been noted that activation of thrombin receptors maybe deleterious. Hauck et al. (1999) Am J Physiol 277:L22-L29.Furthermore, mast cells have also been shown to have thrombin receptors.Cirino et al (1996) J Exp Med 183:821-827. nGPCR-x may also be useful inremodeling of airway structure s in chronic pulmonary inflammation viastimulation of fibroblast procollagen synthesis. See, e.g., Chambers etal. (1998) Biochem J 333:121-127; Trejo et al. (1996) J Biol Chem271:21536-21541.

In another example, increased release of sCD40L and expression of CD40Lby T cells after activation of thrombin receptors suggests that nGPCR-xmay be useful in the treatment of unstable angina due to the role of Tcells and inflammation. See Aukrust et al. (1999) Circulation100:614-620.

A further example is the treatment of inflammatory diseases, such aspsoriasis, inflammatory bowel disease, multiple sclerosis, rheumatoidarthritis, and thyroiditis. Due to the tissue expression profile ofnGPCR-x, inhibition of thrombin receptors may be beneficial for thesediseases. See, e.g., Morris et al. (1996) Ann Rheum Dis 55:841-843. Inaddition to T cells, NK cells and monocytes are also critical cell typeswhich contribute to the pathogenesis of these diseases. See, e.g.,Naldini & Carney (1996) Cell Immunol 172:3542; Hoffman & Cooper (1995)Blood Cells Mol Dis 21:156-167; Colotta et al. (1994) Am J Pathol144:975-985.

Expression of nGPCR-x in bone marrow and spleen may suggest that it mayplay a role in the proliferation of hematopoietic progenitor cells. SeeDiCuccio et al. (1996) Exp Hematol 24:914-918.

As another example, nGPCR-x may be useful in the treatment of acuteand/or traumatic brain injury. Astrocytes have been demonstrated toexpress thrombin receptors. Activation of thrombin receptors may beinvolved in astrogliosis following brain injury. Therefore, inhibitionof receptor activity may be beneficial for limiting neuroinflammation.Scar formation mediated by astrocytes may also be limited by inhibitingthrombin receptors. See, e.g., Pindon et al. (1998) Eur J Biochem255:766-774; Ubl & Reiser. (1997) Glia 21:361-369; Grabham & Cunningham(1995) J Neurochem 64:583-591.

nGPCR-x receptor activation may mediate neuronal and astrocyte apoptosisand prevention of neurite outgrowth. Inhibition would be beneficial inboth chronic and acute brain injury. See, e.g., Donovan et al. (1997) JNeurosci 17:5316-5326; Turgeon et al (1998) J Neurosci 18:6882-6891;Smith-Swintosky et al. (1997) J Neurochem 69:1890-1896; Gill et al.(1998) Brain Res 797:321-327; Suidan et al. (1996) Semin Thromb Hemost22:125-133.

The attached Sequence Listing contains the sequences of thepolynucleotides and polypeptides of the invention and is incorporatedherein by reference in its entirety.

As described above and in Example 4 below, the genes encoding nGPCR-1(nucleic acid sequence SEQ ID NO: 1, SEQ ID NO: 73, amino acid sequenceSEQ ID NO: 2, SEQ ID NO:74), nGPCR-9 (nucleic acid sequence SEQ ID NO:9,SEQ ID NO:77, amino acid sequence SEQ ID NO: 10, SEQ ID NO:78), nGPCR-11(nucleic acid sequence SEQ ID NO:11, SEQ ID NO:79, amino acid sequenceSEQ ID NO:12, SEQ ID NO:80), nGPCR-16 (nucleic acid sequence SEQ ID NO:21, SEQ. ID NO:81, amino acid sequence SEQ ID NO: 22, SEQ ID NO:82),nGPCR-40 (nucleic acid sequence SEQ ID NO:53, SEQ ID NO:83, amino acidsequence SEQ ID NO: 54, SEQ ID NO:84), nGPCR-54 (nucleic acid sequenceSEQ ID NO:59, SEQ ID NO:85, amino acid sequence SEQ ID NO:60, SEQ ID NO:86), nGPCR-56 (nucleic acid sequence SEQ ID NO:63, SEQ ID NO:87, SEQ IDNO:89, amino acid sequence SEQ ID NO:64, SEQ ID NO: 88, SEQ ID NO:90),nGPCR-58 (nucleic acid sequence SEQ ID NO:3, SEQ ID NO: 185, amino acidsequence SEQ ID NO:4, SEQ ID NO: 186) have been detected in brain tissueindicating that these nGPCR-x proteins are neuroreceptors. Theidentification of modulators such as agonists and antagonists istherefore useful for the identification of compounds useful to treatneurological diseases and disorders. Such neurological diseases anddisorders, including but are not limited to, schizophrenia, affectivedisorders, ADHD/ADD (i.e., Attention Deficit-HyperactivityDisorder/Attention Deficit Disorder), and neural disorders such asAlzheimer's disease, Parkinson's disease, migraine, and senile dementiaas well as depression, anxiety, bipolar disease, epilepsy, neuritis,neurasthenia, neuropathy, neuroses, and the like.

Methods of Screening Human Subjects

Thus in yet another embodiment, the invention provides genetic screeningprocedures that entail analyzing a person's genome—in particular theiralleles for GPCRs of the invention—to determine whether the individualpossesses a genetic characteristic found in other individuals that areconsidered to be afflicted with, or at risk for, developing a mentaldisorder or disease of the brain that is suspected of having ahereditary component. For example, in one embodiment, the inventionprovides a method for determining a potential for developing a disorderaffecting the brain in a human subject comprising the steps of analyzingthe coding sequence of one or more GPCR genes from the human subject;and determining development potential for the disorder in said humansubject from the analyzing step.

More particularly, the invention provides a method of screening a humansubject to diagnose a disorder affecting the brain or geneticpredisposition therefor, comprising the steps of: (a) assaying nucleicacid of a human subject to determine a presence or an absence of amutation altering the amino acid sequence, expression, or biologicalactivity of at least one seven transmembrane receptor that is expressedin the brain, wherein the seven transmembrane receptor comprises anamino acid sequence selected from the group consisting of SEQ ID NOS:74, 186, 78, 80, 82, 84, 86, 90, and 94, or an allelic variant thereof,and wherein the nucleic acid corresponds to the gene encoding the seventransmembrane receptor; and (b) diagnosing the disorder orpredisposition from the presence or absence of said mutation, whereinthe presence of a mutation altering the amino acid sequence, expression,or biological activity of allele in the nucleic acid correlates with anincreased risk of developing the disorder. In preferred variations, theseven transmembrane receptor is nGPCR-40 or nGPCR-54 comprising aminoacid sequences set forth in SEQ ID NO: 84 for nGPCR-40 and SEQ ID NO: 86for nGPCR-54, or an allelic variant thereof, and the disease isschizophrenia.

By “human subject” is meant any human being, human embryo, or humanfetus. It will be apparent that methods of the present invention will beof particular interest to individuals that have themselves beendiagnosed with a disorder affecting the brain or have relatives thathave been diagnosed with a disorder affecting the brain.

By “screening for an increased risk” is meant determination of whether agenetic variation exists in the human subject that correlates with agreater likelihood of developing a disorder affecting the brain thanexists for the human population as a whole, or for a relevant racial orethnic human sub-population to which the individual belongs. Bothpositive and negative determinations (i.e., determinations that agenetic predisposition marker is present or is absent) are intended tofall within the scope of screening methods of the invention. Inpreferred embodiments, the presence of a mutation altering the sequenceor expression of at least one nGPCR-40 or nGPCR-54 seven transmembranereceptor allele in the nucleic acid is correlated with an increased riskof developing schizophrenia, whereas the absence of such a mutation isreported as a negative determination.

The “assaying” step of the invention may involve any techniquesavailable for analyzing nucleic acid to determine its characteristics,including but not limited to well-known techniques such as single-strandconformation polymorphism analysis (SSCP) [Orita et al., Proc Natl.Acad. Sci. USA, 86: 2766-2770 (1989)]; heteroduplex analysis [White etal., Genomics, 12: 301-306 (1992)]; denaturing gradient gelelectrophoresis analysis [Fischer et al., Proc. Natl. Acad. Sci. USA,80: 1579-1583 (1983); and Riesner et al., Electrophoresis, 10: 377-389(1989)]; DNA sequencing; RNase cleavage [Myers et al., Science, 230:1242-1246 (1985)]; chemical cleavage of mismatch techniques [Rowley etal., Genomics, 30: 574-582 (1995); and Roberts et al., Nucl. Acids Res.,25: 3377-3378 (1997)]; restriction fragment length polymorphismanalysis; single nucleotide primer extension analysis [Shumaker et al.,Hum. Mutat., 7: 346-354 (1996); and Pastinen et al., Genome Res., 7:606-614 (1997)]; 5′ nuclease assays [Pease et al., Proc. Natl. Acad.Sci. USA, 91:5022-5026 (1994)]; DNA Microchip analysis [Raisay, G.,Nature Biotechnology, 16: 4048 (1999); and Chee et al., U.S. Pat. No.5,837,832]; and ligase chain reaction [Whiteley et al., U.S. Pat. No.5,521,065]. [See generally, Schafer and Hawkins, Nature Biotechnology,16: 33-39 (1998).] All of the foregoing documents are herebyincorporated by reference in their entirety.

Thus, in one preferred embodiment involving screening nGPCR-40 ornGPCR-54 sequences, for example, the assaying step comprises at leastone procedure selected from the group consisting of: (a) determining anucleotide sequence of at least one codon of at least one nGPCR-40 ornGPCR-54 allele of the human subject; (b) performing a hybridizationassay to determine whether nucleic acid from the human subject has anucleotide sequence identical to or different from one or more referencesequences; (c) performing a polynucleotide migration assay to determinewhether nucleic acid from the human subject has a nucleotide sequenceidentical to or different from one or more reference sequences; and (d)performing a restriction endonuclease digestion to determine whethernucleic acid from the human subject has a nucleotide sequence identicalto or different from one or more reference sequences.

In a highly preferred embodiment, the assaying involves sequencing ofnucleic acid to determine nucleotide sequence thereof, using anyavailable sequencing technique. [See, e.g., Sanger et al., Proc. Natl.Acad. Sci. (USA), 74: 5463-5467 (1977) (dideoxy chain terminationmethod); Mirzabekov, TIBTECH, 12: 27-32 (1994) (sequencing byhybridization); Drmanac et al., Nature Biotechnology, 16: 54-58 (1998);U.S. Pat. No. 5,202,231; and Science, 260: 1649-1652 (1993) (sequencingby hybridization); Kieleczawa et al., Science, 258: 1787-1791 (1992)(sequencing by primer walking); (Douglas et al., Biotechniques, 14:824828 (1993) (Direct sequencing of PCR products); and Akane et al.,Biotechniques 16: 238-241 (1994); Maxam and Gilbert, Meth. Enzymol., 65:499-560 (1977) (chemical termination sequencing), all incorporatedherein by reference.] The analysis may entail sequencing of the entirenGPCR gene genomic DNA sequence, or portions thereof, or sequencing ofthe entire seven transmembrane receptor coding sequence or portionsthereof. In some circumstances, the analysis may involve a determinationof whether an individual possesses a particular allelic variant, inwhich case sequencing of only a small portion of nucleic acid—enough todetermine the sequence of a particular codon characterizing the allelicvariant—is sufficient This approach is appropriate, for example, whenassaying to determine whether one family member inherited the sameallelic variant that has been previously characterized for anotherfamily member, or, more generally, whether a person's genome contains anallelic variant that has been previously characterized and correlatedwith a mental disorder having a heritable component.

In another highly preferred embodiment, the assaying step comprisesperforming a hybridization assay to determine whether nucleic acid fromthe human subject has a nucleotide sequence identical to or differentfrom one or more reference sequences. In a preferred embodiment, thehybridization involves a determination of whether nucleic acid derivedfrom the human subject will hybridize with one or more oligonucleotides,wherein the oligonucleotides have nucleotide sequences that correspondidentically to a portion of the GPCR gene sequence taught herein, suchas the nGPCR-40 or nGPCR-54 coding sequence set forth in SEQ ID NOS: 83for nGPCR-40 or 85 for nGPCR-54, or that correspond identically exceptfor one mismatch. The hybridization conditions are selected todifferentiate between perfect sequence complementarity and imperfectmatches differing by one or more bases. Such hybridization experimentsthereby can provide single nucleotide polymorphism sequence informationabout the nucleic acid from the human subject, by virtue of knowing thesequences of the oligonucleotides used in the experiments.

Several of the techniques outlined above involve an analysis wherein oneperforms a polynucleotide migration assay, e.g., on a polyacrylamideelectrophoresis gel (or in a capillary electrophoresis system), underdenaturing or non-denaturing conditions. Nucleic acid derived from thehuman subject is subjected to gel electrophoresis, usually adjacent to(or co-loaded with) one or more reference nucleic acids, such asreference GPCR-encoding sequences having a coding sequence identical toall or a portion of SEQ ID NOS: 83 or 85 (or identical except for oneknown polymorphism). The nucleic acid from the human subject and thereference sequence(s) are subjected to similar chemical or enzymatictreatments and then electrophoresed under conditions whereby thepolynucleotides will show a differential migration pattern, unless theycontain identical sequences. [See generally Ausubel et al. (eds.),Current Protocols in Molecular Biology, New York: John Wiley & Sons,Inc. (1987-1999); and Sambrook et al., (eds.), Molecular Cloning, ALaboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1989), both incorporated herein by reference in theirentirety.]

In the context of assaying, the term “nucleic acid of a human subject”is intended to include nucleic acid obtained directly from the humansubject (e.g., DNA or RNA obtained from a biological sample such as ablood, tissue, or other cell or fluid sample); and also nucleic acidderived from nucleic acid obtained directly from the human subject. Byway of non-limiting examples, well known procedures exist for creatingcDNA that is complementary to RNA derived from a biological sample froma human subject, and for amplifying (e.g., via polymerase chain reaction(PCR)) DNA or RNA derived from a biological sample obtained from a humansubject. Any such derived polynucleotide which retains relevantnucleotide sequence information of the human subject's own DNA/RNA isintended to fall within the definition of “nucleic acid of a humansubject” for the purposes of the present invention.

In the context of assaying, the term “mutation” includes addition,deletion, and/or substitution of one or more nucleotides in the GPCRgene sequence (e.g., as compared to the seven transmembranereceptor-encoding sequences set forth of SEQ ID NOS: 74, 186, 78, 80,82, 84, 86, 90, and 94) and other polymorphisms that occur in introns(where introns exist) and that are identifiable via sequencing,restriction fragment length polymorphism, or other techniques. Thevarious activity examples provided herein permit determination ofwhether a mutation modulates activity of the relevant receptor in thepresence or absence of various test substances.

In a related embodiment, the invention provides methods of screening aperson's genotype with respect to GPCR's of the invention, andcorrelating such genotypes with diagnoses for disease or withpredisposition for disease (for genetic counseling). For example, theinvention provides a method of screening for an nGPCR-40 or nGPCR-54hereditary schizophrenia genotype in a human patient, comprising thesteps of: (a) providing a biological sample comprising nucleic acid fromthe patient, the nucleic acid including sequences corresponding to saidpatients nGPCR-40 or nGPCR-54 alleles; (b) analyzing the nucleic acidfor the presence of a mutation or mutations; (c) determining an nGPCR-40or nGPCR-54 genotype from the analyzing step; and (d) correlating thepresence of a mutation in an nGPCR-40 or nGPCR-54 allele with ahereditary schizophrenia genotype. In a preferred embodiment, thebiological sample is a cell sample containing human cells that containgenomic DNA of the human subject. The analyzing can be performedanalogously to the assaying described in preceding paragraphs. Forexample, the analyzing comprises sequencing a portion of the nucleicacid (e.g., DNA or RNA), the portion comprising at least one codon ofthe nGPCR-40 or nGPCR-54 alleles.

Although more time consuming and expensive than methods involvingnucleic acid analysis, the invention also may be practiced by assayingprotein of a human subject to determine the presence or absence of anamino acid sequence variation in GPCR protein from the human subject.Such protein analyses may be performed, e.g., by fragmenting GPCRprotein via chemical or enzymatic methods and sequencing the resultantpeptides; or by Western analyses using an antibody having specificityfor a particular allelic variant of the GPCR.

The invention also provides materials that are useful for performingmethods of the invention. For example, the present invention providesoligonucleotides useful as probes in the many analyzing techniquesdescribed above. In general, such oligonucleotide probes comprise 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 nucleotides that have a sequence that isidentical, or exactly complementary, to a portion of a human GPCR genesequence taught herein (or allelic variant thereof), or that isidentical or exactly complementary except for one nucleotidesubstitution. In a preferred embodiment, the oligonucleotides have asequence that corresponds in the foregoing manner to a human GPCR codingsequence taught herein, and in particular, the coding sequences setforth in SEQ ID NO: 83 and 85. In one variation, an oligonucleotideprobe of the invention is purified and isolated. In another variation,the oligonucleotide probe is labeled, e.g., with a radioisotope,chromophore, or fluorophore. In yet another variation, the probe iscovalently attached to a solid support. [See generally Ausubel et al.And Sambrook et al., supra.]

In a related embodiment, the invention provides kits comprising reagentsthat are useful for practicing methods of the invention. For example,the invention provides a kit for screening a human subject to diagnoseschizophrenia or a genetic predisposition therefor, comprising, inassociation: (a) an oligonucleotide useful as a probe for identifyingpolymorphisms in a human nGPCR-40 or nGPCR-54 seven transmembranereceptor gene, the oligonucleotide comprising 6-50 nucleotides that havea sequence that is identical or exactly complementary to a portion of ahuman nGPCR-40 or nGPCR-54 gene sequence or nGPCR-40 or nGPCR-54 codingsequence, except for one sequence difference selected from the groupconsisting of a nucleotide addition, a nucleotide deletion, ornucleotide substitution; and (b) a media packaged with theoligonucleotide containing information identifying polymorphismsidentifiable with the probe that correlate with schizophrenia or agenetic predisposition therefor. Exemplary information-containing mediainclude printed paper package inserts or packaging labels; and magneticand optical storage media that are readable by computers or machinesused by practitioners who perform genetic screening and counselingservices. The practitioner uses the information provided in the media tocorrelate the results of the analysis with the oligonucleotide with adiagnosis. In a preferred variation, the oligonucleotide is labeled.

In still another embodiment, the invention provides methods ofidentifying those allelic variants of GPCRs of the invention thatcorrelate with mental disorders. For example, the invention provides amethod of identifying a seven transmembrane allelic variant thatcorrelates with a mental disorder, comprising steps of: (a) providing abiological sample comprising nucleic acid from a human patient diagnosedwith a mental disorder, or from the patient's genetic progenitors orprogeny; (b) analyzing the nucleic acid for the presence of a mutationor mutations in at least one seven transmembrane receptor that isexpressed in the brain, wherein the at least one seven transmembranereceptor comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 74, 186, 78, 80, 82, 84, 86, 90, and 94 or anallelic variant thereof, and wherein the nucleic acid includes sequencecorresponding to the gene or genes encoding the at least one seventransmembrane receptor, (c) determining a genotype for the patient forthe at least one seven transmembrane receptor from said analyzing step;and (d) identifying an allelic variant that correlates with the mentaldisorder from the determining step. To expedite this process, it may bedesirable to perform linkage studies in the patients (and possibly theirfamilies) to correlate chromosomal markers with disease states. Thechromosomal localization data provided herein facilitates identifying aninvolved GPCR with a chromosomal marker.

The foregoing method can be performed to correlate GPCR's of theinvention to a number of disorders having hereditary components that arecausative or that predispose persons to the disorder. For example, inone preferred variation, the disorder is schizophrenia, and the at leastone seven transmembrane receptor comprises nGPCR-40 having an amino acidsequence set forth in SEQ ID NO: 84 or an allelic variant thereof.

Also contemplated as part of the invention are polynucleotides thatcomprise the allelic variant sequences identified by such methods, andpolypeptides encoded by the allelic variant sequences, andoligonucleotide and oligopeptide fragments thereof that embody themutations that have been identified. Such materials are useful in invitro cell-free and cell-based assays for identifying lead compounds andtherapeutics for treatment of the disorders. For example, the variantsare used in activity assays, binding assays, and assays to screen foractivity modulators described herein. In one preferred embodiment, theinvention provides a purified and isolated polynucleotide comprising anucleotide sequence encoding a nGPCR-40 or nGPCR-54 receptor allelicvariant identified according to the methods described above; and anoligonucleotide that comprises the sequences that differentiate theallelic variant from the nGPCR-40 or nGPCR-54 sequences set forth in SEQID NOS: 83 and 88. The invention also provides a vector comprising thepolynucleotide (preferably an expression vector); and a host celltransformed or transfected with the polynucleotide or vector. Theinvention also provides an isolated cell line that is expressing theallelic variant GPCR polypeptide; purified cell membranes from suchcells; purified polypeptide; and synthetic peptides that embody theallelic variation amino acid sequence. In one particular embodiment, theinvention provides a purified polynucleotide comprising a nucleotidesequence encoding a nGPCR-40 seven transmembrane receptor protein of ahuman that is affected with schizophrenia; wherein said polynucleotidehybridizes to the complement of SEQ ID NO: 83 under the followinghybridization conditions: (a) hybridization for 16 hours at 42° C. in ahybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10%dextran sulfate and (b) washing 2 times for 30 minutes at 60° C. in awash solution comprising 0.1×SSC and 1% SDS; and wherein thepolynucleotide encodes a nGPCR-40 amino acid sequence that differs fromSEQ ID NO: 84 by at least one residue.

An exemplary assay for using the allelic variants is a method foridentifying a modulator of nGPCR-x biological activity, comprising thesteps of: (a) contacting a cell expressing the allelic variant in thepresence and in the absence of a putative modulator compound; (b)measuring nGPCR-x biological activity in the cell; and (c) identifying aputative modulator compound in view of decreased or increased nGPCR-xbiological activity in the presence versus absence of the putativemodulator.

Additional features of the invention will be apparent from the followingExamples. Examples 1, 2, 4, 11, 12, and 13 are actual, while theremaining Examples are prophetic. Additional features and variations ofthe invention will be apparent to those skilled in the art from theentirety of this application, including the detailed description, andall such features are intended as aspects of the invention. Likewise,features of the invention described herein can be re-combined intoadditional embodiments that also are intended as aspects of theinvention, irrespective of whether the combination of features isspecifically mentioned above as an aspect or embodiment of theinvention. Also, only such limitations which are described herein ascritical to the invention should be viewed as such; variations of theinvention lacking limitations which have not been described herein ascritical are intended as aspects of the invention.

EXAMPLES Example 1 Identification of nGPCR-X

A. Database Search

The Celera database was searched using known GPCR receptors as querysequences to find patterns suggestive of novel G protein-coupledreceptors. Positive hits were further analyzed with the GCG programBLAST to determine which ones were the most likely candidates to encodeG protein-coupled receptors, using the standard (default) alignmentproduced by BLAST as a guide.

Briefly, the BLAST algorithm, which stands for Basic Local AlignmentSearch Tool is suitable for determining sequence similarity (Altschul etal., J. Molec. Biol., 1990, 215, 403-410, which is incorporated hereinby reference in its entirety). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). This algorithm involvesfirst identifying high scoring sequence pair (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to find HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Extension for the word hits in each direction are haltedwhen: 1) the cumulative alignment score falls off by the quantity X fromits maximum achieved value; 2) the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or 3) the end of either sequence is reached. The Blastalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The Blast program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad.Sci. USA, 1992, 89, 10915-10919, which is incorporated herein byreference in its entirety) alignments (13) of 50, expectation (E) of 10,M=5, N=4, and a comparison of both strands.

The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993,90, 5873-5787, which is incorporated herein by reference in itsentirety) and Gapped BLAST perform a statistical analysis of thesimilarity between two sequences. One measure of similarity provided bythe BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a GPCR gene or cDNA if thesmallest sum probability in comparison of the test nucleic acid to aGPCR nucleic acid is less than about 1, preferably less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

Homology searches were performed with the program BLAST version 2.08. Acollection of 340 query amino acid sequences derived from GPCR's wasused to search the genomic DNA sequence using TBLASTN and alignmentswith an E-value lower than 0.01 were collected from each BLAST search.The amino acid sequences have been edited to remove regions in thesequence that produce non-significant alignments with proteins that arenot related to GPCR's.

Multiple query sequences may have a significant alignment to the samegenomic region, although each alignment may not cover exactly the sameDNA region. A procedure is used to determine the region of maximumcommon overlap between the alignments from several query sequences. Thisregion is called the consensus DNA region. The procedure for determiningthis consensus involves the automatic parsing of the BLAST output filesusing the program MSPcrunch to produce a tabular report. From thistabular report the start and end of each alignment in the genomic DNA isextracted. This information was used by a PERL script to derive themaximum common overlap. These regions were reported in the form of aunique sequence identifier, a start and the end position in thesequence. The sequences defined by these regions were extracted from theoriginal genomic sequence file using the program fetchdb.

The consensus regions were assembled into a non-redundant set by usingthe program phrap. After assembly with phrap a set of contigs andsingletons was defined as candidate DNA regions coding for nGPCR-x.These sequences were then submitted for further sequence analysis.

Further sequence analysis involved the removal of sequences previouslyisolated and removal of sequences related to olfactory GPCRs. Thetransmembrane regions for the sequences that remained were determinedusing a FORTRAN computer program called “tmtrest.all” [Parodi et al.,Comput. Appl. Biosci. 5:527-535(1994)]. Only sequences that containedtransmembrane regions in a pattern found in GPCRs were retained.

cDNAs were sequenced directly using an ABI377 fluorescence-basedsequencer (Perkin-Elmer/Applied Biosystems Division, PE/ABD, FosterCity, Calif.) and the ABI PRISM™ Ready Dye-Deoxy Terminator kit with TaqFS™ polymerase. Each ABI cycle sequencing reaction contained about 0.5μg of plasmid DNA. Cycle-sequencing was performed using an initialdenaturation at 98° C. for 1 minute, followed by 50 cycles using thefollowing parameters: 98° C. for 30 seconds, annealing at 50° C. for 30seconds, and extension at 60° C. for 4 minutes. Temperature cycles andtimes were controlled by a Perkin-Elmer 9600 thermocycler. Extensionproducts were purified using Centriflex™ gel filtration cartridges(Advanced Genetic Technologies Corp., Gaithersburg, Md.). Each reactionproduct was loaded by pipette onto the column, which is then centrifugedin a swinging bucket centrifuge (Sorvall model RT6000B tabletopcentrifuge) at 1500×g for 4 minutes at room temperature. Column-purifiedsamples were dried under vacuum for about 40 minutes and then dissolvedin 5 μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA,and 1.6 mg/ml Blue Dextran). The samples were then heated to 90° C. forthree minutes and loaded into the gel sample wells for sequence analysisusing the ABI377 sequencer. Sequence analysis was performed by importingABI377 files into the Sequencer program (Gene Codes, Ann Arbor, Mich.).Generally, sequence reads of 700 bp were obtained. Potential sequencingerrors were minimized by obtaining sequence information from both DNAstrands and by re-sequencing difficult areas using primers annealing atdifferent locations until all sequencing ambiguities were removed.

The following Table 5 contains the sequences of the polynucleotides andpolypeptides of the invention. Start and stop codons within thepolynucleotide sequence are identified by boldface type. Thetransmembrane domains within the polypeptide sequence are identified byunderlining. TABLE 5 The following DNA sequence beGPCR-seq1 <SEQ ID NO.1> was identified in H. sapiens:GTCTGGGGGTGGGGGATGCTGGGACAGGGGTCAATTGCCTGAAGCAAGTGCTCTCATCCCCCTAGCTCCTGCTGATCTAGTTGGGGCTCCAGAGTGGGGAGGAGAAAGGCACTTTGAAACTTCTCTGCCCTTACCGTCTTAGCCATCAAACTCTGAGCTGGAGATAGTGACGATGTGACAGGAACTTTCCCTGGGCCTCTCTGGGCCACAATTCCTGGCCGAGAGAAAGAGGAGGAATGAGGTGAGCACCTTCTTCACTCCTAGGGCCATGTGGTAGAGCTGCAGTCGCACCTCCTTCTGCCAATAGGCATAGATGAGTGGGTTGAGCAGGGAGTTGCCCACGCCGAGCAGCCACAGGTACCGTTCCAGCACTAGGTAGAGGTGACACTCCTGGCAGGCCACCTGCACAATGCCAGTGATAAGGAAGGGGGTCCAGGATAGAGCAAAGCTCCCAATGAGAACAGACACAGTACGGAGAGCTTTGAAGTCGCTGGGAGTCCGTGGGGATCGATAACCTCCAGCCATGGCTCCTGCATGTTCCATCTTTCGAATCTGCTGGCTGTGCATGGAGGCAATTTGAGCATGTCGCAGTAGAAGAAGACAAAGAGGAGCATGGCTGGGAAGAAGCCAACGCAGGAGAGGGTCAGCACGAAGTGAGGGTGAAATACAGCAAAGAAGCTGCACTGCCCTTTGTAGGCAGTCTGCTGGAACATGGGGATTCCGAGTGGGAGGAAGCCAATGAGGTAAGACACTAACCACAGCCCGGCAATGCAGGCCCCGGCCACGAACCCACTCATGATCTTCAAGTAGCGGAAGGGCTGCTTGATGGCAAGGTACCTGTCAAAGGTGATCAGCATGACCGTGAGGACAGAGGCAGCTGCGGAGGAAGTGACAAATGCCATCCGCAGGCTGCACAGGGTCTTCTGTGTGGGCCGAGAAGGGCTGGAGAGCTGGTCTGTGAGTAGGCCAGAGATGGCCACACCAATCAAGGTGTCAGCCACAGCCAGATTCAAGGTGAAGCAGAGACTGACACCATCATTCTTGTGGATCAACAGCAGCACAGCCACAGCCACTAGTGTGTTAGTAGCAATGATGAGGGAGGCCAGGACAGCAAGGATCACTCCAAATGAGAAAGATGATTCCATGTCTCGAAGTGGCAGGACTTCACTTACCAGGGCATG The following amino acid sequence <SEQ IDNO. 2> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 1:MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPTQKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLGIPMFQQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPSDFKALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLYHMALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG The following DNA sequencebeGPCR-seq3 <SEQ ID NO. 3> was identified in H. sapiens:CAGCGCGAGCGCCTTCATGGTGACGGTGTCCATGCGCTGGCAGTGTCTGCGTGCCACCCGGTGCACCTGGAGCGAGGTGAGGCAGAGCACCGCCAGCGGCAGCACGAAGCCCACGGCATGGAGCGTGGCGGTGAAGGCTGCGAAGCGCGGACGCTCAGGCTCGGGCGGCAGGCGCAGCGAACAGGACGCGAAGGCGCTGCTGTAGCCAAGCCACGAGCAGCCAAGTGCAGCGCCTGAGAAGGCCAGCGACTGTCCCCAGGCACAGCCCAGCAGCAGGCCGGCATAGCGCGGTCGCAGGCGTCCGGCGTAGCGCAGTGGGAAGCCCACTGCCAGCCACTGGTCTGCGCTCAGCGCCGCCACGCTCAGCGCCGCGTTGGACGCCAGGAAGGTGTCCAGGAAGCCAATGACTTGGCATGCGCCGGGCGCCGACGGTGTCCGCCCGCGCATCACACCGAGCAGCGTGAAGGGCATGTCCAGCGCCGCCAGCAGCAGGTGGCCCAGAGACAGATTCACCAGGAGGACGCCTGAGGCTCGAGTGCGGAGCTCAGCGCTGTAGGCGCAACAAAGCAGCACCAGTGCGTTGGATAGCAGCGCCACGGCCAGTACCATCACCAGGAGACCCGCCAGCAGCGCCTCGCCGGGGCCCATGGCGCTAGC The following amino acid sequence <SEQ ID NO. 4> is thepredicted amino acid sequence derived from the DNA sequence of SEQ IDNO. 3:SAMGPGEALLAGLLVMVLAVALLSNALVLLCCAYSAELRTRASGVLLVNLSLGHLLLAALDMPFTLLGVMRGRTPSAPGACQVIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPRYAGLLLGCAWGQSLAFSGAALGCSWLGYSSAFASCSLRLPPEPERPRFAAFTATLHAVGFVLPLAVLCLTSLQVHRVARRHCQRMDTVTMKALAThe following DNA sequence beGPCR-seq4 <SEQ ID NO. 5> was identified inH. sapiens:TGTGCAGGTGTGATCTCCATTCCTTTGTACATCCCTCACACGCTGTTCGATGGGATTTTGGAAAGGAAATCTGTGTATTTTGGCTCACTACTGACTATCTGTTATGTACAGCATCTGTATATAACATTGTCCTCATCAGCTATGATCGATACCTGTCAGTCTCAAATGCTGTAAGTCGAACACATTAATTTATCCCCCTTAGAAGATTATGTAAATGTATA The following amino acid sequence <SEQ ID NO. 6> is the predictedamino acid sequence derived from the DNA sequence of SEQ ID NO. 5:CAGVISIPLYIPHTLFEWDFGKEICVFWLTTDYLLCTASVYNIVLISYDRYLSVSNAVSRTHFIPLRRLCKCI The following DNA sequence beGPCR-seq5 <SEQ ID NO. 7> wasidentified in H. sapiens:GACGTCGAAGCAGGTGATGATGCCCAGGGCGTGCACCGGGTAGGTGAGATCGGTGCGCGCCAGCGGGGACAGGGCGGTCAGGAGCAGCAGCCAGGTCCCTGCACACGCGGCCACCGCGTAACGACGGCGGCGCCAGCGCTTGGAGCTGAGCGGGTACAGGATCCCCAGGAAGCGCTCCACGCTGATACAGGTCATGGTGAGGATGCTGGAATACATGTTTGCGTAAAAGGCCACGGTCACCACGTTGCAAAGCAGCACCCCGAATACCCAGTGGTGGCGGTTGCAATGGTAGTAGATTTGGAAAGGCAACACGCTGGCCAGCATCAGGTCCGTGACGCTCAGGTTGATCATGAAGATGACCGACGGGGATCTGGGCCCCATGCGCCGGCACAGCACCCACAGAGAGAAGAGGTTGCCCGGGATGCTGACCGCCGCCACCAGCGAGTACACCACGGGCAGGGCCACCGCGATCGCCGGGTTCCGCAGCATCTGCAGCGTCGCGTTGTCThe following amino acid sequence <SEQ ID NO. 8> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 7:DNATLQMLRNPAIAVALPVVYSLVAAVSIPGNLFSLWVLCRRMGPRSPSVIFMINLSVTDLMLASVLPFQIYYHCNRHHWVFGVLCNLVVTVAFYANMYSSILTMTCISVERFLGILYPLSSKRWRRRRYAVAACAGTWLLLLTALSPLARTDLTYPVHALGIITCFDV The following DNA sequence beGPCR-seq9 <SEQ IDNO. 9> was identified in H. sapiens:CCCATGTTCCTGCTCCTGGGCAGCCTCACGTTGTCGGATCTGCTGGCAGGCGCCGCCTACGCCGCCAACATCCTACTGTCGGGGCCGCTCACGCTGAAACTGTCCCCCGCGCTCTGGTTCGCACGGGAGGGAGGCGTCTTCGTGGCACTCACTGCGTCCGTGCTGAGCCTCCTGGGCATCGCGCTGGAGCGCAGCCTCACCATGGCGCGCAGGGGGCCCGCGCCCGTCTCCAGTCGGGGGCGCACGCTGGCGATGGCAGCCGCGGCCTGG The followingamino acid sequence <SEQ ID NO. 10> is the predicted amino acid sequencederived from the DNA sequence of SEQ ID NO. 9:PMFLLLGSLTLSDLLAGAAYAANILLSGPLTLKLSPALWFAREGGVFVALTASVLSLLGIALERSLTMARRGPAPVSSRGRTLAMAAAAW The following DNA sequence beGPCR-seq11 <SEQ ID NO.11> was identified in H. sapiens:CTGCTCATTGTGGCCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAGCCCAGCACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCCTGCCTTTTCGGACAGACTATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGGCTCTTCACGTTGGCCATGAACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTTCAAAGTGGTCCACCCCCACCACGCGGTGAACACTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCCTGTGGGCCCTGGTCATCCTGGGAACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCCGTCTCCTGTGAGAGCTTCATCATGGAGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTTTATGCCCCTCGGCATCATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGCTGGCCAGACAGGCTCGGATGAAGAAGGCGACCCGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCTACCTGCCCAGCGTGTCTGCTAGACTCTATTTCCTCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCATGGGGCCCTGCACATAACCCTCAGCTTCACCTACATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTTCAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTCAAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACACTCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCG The following amino acid sequence<SEQ ID NO. 12> is the predicted amino acid sequence derived from theDNA sequence of SEQ ID NO. 11:LLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTDYYLRRRHWAFGDIPCRVGLFTLAMNRAGSIVFLTVVAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILGTVYLLLENHLCVQETAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKATRFIMVVAIVFITCYLPSVSARLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKLKICSLKPKQPGHSKTQRPEEMPIS The following DNA sequence beGPCR-seq12 <SEQ ID NO. 13> wasidentified in H. sapiens:TGGAGCTGTGCCACCACCTATCTGGTGAACCTGATGGTGGCCGACCTGCTTTATGTGCTATTGCCCTTCCTCATCATCACCTACTCACTAGATGACAGGTGGCCCTTCGGGGAGCTGCTCTGCAAGCTGGTGCACTTCCTGTTCTATATCAACCTTTACGGCAGCATCCTGCTGCTGACCTGCATCTCTGTGCACCAGTTCCTAGGTGTGTGCCACCCACTGTGTTCGCTGCCCTACCGGACCCGCAGGCATGCCTGGCTGGGCACCAGCACCACCTGGGCCCTGGTGGTCCTCCAGCTGCTGCCCACACTGGCCTTCTCCCACACGGACTACATCAATGGCCAGATGATCTGGTATGACATGACCAGCCAAGAGAATTTTGATCGGCTTTTTGCCTACGGCATAGTTCTGACATTGTCTGGCTTTCTTTCCCTCCTTGGTCATTTTGGTGTGCTATTCACTGATGGTCAGGAGCCTGATCAAGCCAGAGGAGAACCTCATGAGGACAGG The following amino acid sequence <SEQ ID NO. 14> is thepredicted amino acid sequence derived from the DNA sequence of SEQ IDNO. 13:WSCATTYLVNLMVADLLYVLLPFLIITYSLDDRWPFGELLCKLVHFLFYINLYGSILLLTCISVHQFLGVCHPLCSLPYRTRRHAWLGTSTTWALVVLQLLPTLAFSHTDYINGQMIWYDMTSQENFDRLFAYGIVLTLSGFLSLLGHFGVLFTDGQEPDQARGEPHEDR The following DNA sequence beGPCR-seq14 <SEQ IDNO. 15> was identified in H. sapiens:CCACCACGCGCAGCACGCCGACAGGGCCTCTCCCTCCCATTCTCCCGCAGGCCCGGACGACCACGCTGCCTCCAGCCGGTCGGCAAACTAGGGCAGCTCGCAGCCCACGAACAGCAGCCCCAGCAGCTGGCTCATCTTCAGGCTCTGCACCTTGGCGCGGGGCATCGCGCTGGGCGCACGGGCTCCACCTGGGCTCGCCGACCAGGCCGCTGCACCCGCTGGGGCCTTCAGCCGGTGCCGCCACCAGACGGAGAGTAGGTGGCCACAAGCGACACCCATGATCTTAACAGGCGCGACGAAGCCCGCGACGGCCTCATAGAACGCGTACACCTGCACGTGCCAGCGCTGCAGGAGCGCGAAGATCCAGTGGCAGCGACGCATCCCCGGCCAGGCTCGGGCGGAGAGTGGCGCGCCTGGCTGCAGAGACGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGTACTAGCGCACCACAAACCCCGACCCCCGCGCCAGCAGCAGTGCCAGCAGCCAGCCCAGGGCGGCGAGGGCACGCGCGGGCAGCGGCCGGCCGTGCGGAAGACGCACCGCGCGCCGGCGCTCGAGGGCGATGAGCACCACGAGGTGGGCCGAGGCGCCCCGCCCGGATGCCTGCAGCAGCTGCAGGAAGCGGCACGCCAGGTCCCCCGTGGCCGCGCGGGGCTCGCCCAGCAGTTCCCAGGCCAGCTGTGACAGCGCCGTGCCCCCGCACGCGTACAGGTCCGCCAGGGCCAGCTGCACCAGCAGGAAGTCCATCTTGCGACGCTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACAGGCGGCACAGCACTGTGGTGTTGCCTGCCACCGCCACCACCAGGATGACCCCCAGGAACACCAGGCGGACGCG The following amino acidsequence <SEQ ID NO. 16> is the predicted amino acid sequence derivedfrom the DNA sequence of SEQ ID NO. 15:RVRLVFLGVILVVAVAGNTTVLCRLXXXXXXXXXXKRRKMDFLLVQLALADLYACGGTALSQLAWELLGEPRAATGDLACRFLQLLQASGRGASAHLVVLIALERRRAVRLPHGRPLPARALAALGWLLALLLARGSGFVVRYXXXXXXXXXXXTSLQPGAPLSARAWPGMRRCHWIFALLQRWHVQVYAFYEAVAGFVAPVKIMGVACGHLLSVWWRHRLKAPAGAAAWSASPGGARAPSAMPRAKVQSLKMSQLLGLLFVGCELPFADRLEAAWSSGPAGEWEGEALSACCAWW The following DNA sequence beGPCR-seq15 <SEQ ID NO. 17> wasidentified in H. sapiens:TCTAAGTTTTTCTCTGAACTTTGAGCCTGTGAAAAAAGAAGGGATGCTGCCTCAGGCCACCCCAGCCTAGATACTCACTCTGAGTGCCATGAGGTAGTAGAGGACACTGATGACAGTCATGGGGAGGAGGTAGAATAGGAAGGAGGTGACCTGGATGATGAAATTGTAGATCCACATGGGCTTGATGACCGTACAGGTGGCCGAACCTGGGACCAGGGACCCATTGGGGAAGTAGTGGAACTTGATGCCATGGATGCTGGTGTTGGGCAGGGAGAAGAGCACGGAGAAGCCCCAGACGATGCCGAGGATCCTGAGGGCCCGGCGCCGGGTGCTCTGCAGTTTGGCGCGGAACGGGTGTAGGATGGCCACGTAGCGCTCCACGCTGACGGTGGTGATGCTGAGGATGGAGGCGAAGCACACGGTCTCAAAGAGGGCCGTCTTGAAGTAGCAGCCCACGGGCCCGAACAAGAAAGGGTAGTTGCGCCACATCTCATAGACCTCCAGGGGCATTCCAAGGAGCAGGACCAGGAGGTCAGAGACCGCCAGGCTGAAGAGGTAGTAGTTGGTGGGCGTCTTCATAGCCTGGTGCTGCAGAATCACCAGGCACACCAGGACATTGCCAATGACCCCCACCACAAAAATTGGCACATACACCACAGACACGGGGAGGAAGAAGTGGCTGCGCCGAGGTCCGCAGAGGAAGGCCAGATACTCCTCGGTGCTGTTCAGGTGTTTCTGGAATGGATCTTCTAGTTTCTGCTGGTAGATCCAGGAAGCATTCTGAAGTTTTTCCATCCCTGA The following amino acid sequence <SEQ ID NO. 18>is the predicted amino acid sequence derived from the DNA sequence ofSEQ ID NO. 17:SGMEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQAMKTPNTYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVAILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVTSFLFYLLPMTVISVLYYLMALRVSIAGVAG The following DNA sequence beGPCR-seq18<SEQ ID NO. 19> was identified in H. sapiens:ATCAAGATGATTTTTGCTATCGTGCAAATTATTGGATTTTCCAACTCCATCTGTAATCCCATTGTCTATGCATTTATGAATGAAAACTTCAAAAAAAATGTTTTGTCTGCAGTTTGTTATTGCATAGTAAATAAAACCTTCTCTCCAGCACAAAGGCATGGAAATTCAGGAATTACAATGATGCGGAAGAAAGCAAAGTTTTCCCTCAGAGAGAATCCAGTG The following amino acid sequence <SEQ ID NO. 20> is thepredicted amino acid sequence derived from the DNA sequence of SEQ IDNO. 19:IKMIFAIVQIIGFSNSICNPIVYAFMNENFKKNVLSAVCYCIVNKTFSPAQRHGNSGITMMRKKAKFSLRENPThe following DNA sequence beGPCR-seq16 <SEQ ID NO. 21> was identifiedin H. sapiens:GCCACAGCATGCAGTTTTCTGTAGAATTCCACTTTGTCTTTGCACTTGAAGAAGATGAGGTATCTGGTGACCAGGATCACCACATAGAATAGGAACCGTGAGGTACATGTGGATGTGCAGCATGGCACTCACAAATTTGCAGAAGGGCAGCCCAAACATCCAAGTCTTCTTGATGAGGTAGGTCAAGCGAAATGGCACTGTCAGCAGAAAAACGCTGTGGACCACCACCAAGTTAATGACCGCCATGGTGGTCACTGACCGGGTGTTCATTTTCACCAGGAGGAAAAGAATGGAAATGACACCCACCAGCCCGCCAATAAGCACTATGAAGTAGAGGCTGATTAAGTGGGGTGTCACTATAGGATCGCAAGAGGAATTCCTGGAGGTATTGTGGCCAGGCATACTTGGGAAGTCACCTGGAGGAGAAAAAGCACCAGAGTAACTGAC The following amino acid sequence <SEQ ID NO. 22>is the predicted amino acid sequence derived from the DNA sequence ofSEQ ID NO. 21:VSYSGAFSPPGDFPSMPGHNTSRNSSCDPIVTPHLISLYFIVLIGGLVGVISILFLLVKMNTRSVTTMAVINLVVVHSVFLLTVPFRLTYLIKKTWMFGLPFCKFVSAMLHIHMYLTVPILCGDPGHQIPHLLQVQRQSGILQKTACCG The following DNA sequence beGPCR-seq17 <SEQ ID NO. 23> wasidentified in H. sapiens:ACTGACCAAGGTCAGGGCATCGACTGAGGCTAGAAGGCCACAGGAAATGCCAGTCAAGGTGTTGGCGCCTGCAATCGCACCTACCACAAACTTGACCGGGGGCAGGGGGGCAGGCCCGCCAGCGAACACGGTCAGCAGCACCAGTCCATTGCAGAGCACGGAGAGCAACACGATGGCCCACACGGCCAGGCGGATGCCCCAGCTTTCAAAGAGGTACTCACA The following amino acid sequence <SEQ ID NO. 24> is thepredicted amino acid sequence derived from the DNA sequence of SEQ IDNO. 23:CEYLFESWGIRLAVWAIVLLSVLCNGLVLLTVFAGGPAPLPPVKFVVGAIAGANTLTGISCGLLASVDALTLVS The following DNA sequence beGPCR-seq20 <SEQ ID NO. 25> was identifiedin H. sapiens:AACCCCATCATCTACACGCTCACCAACCGCGACCTGCGCCACGCGCTCCTGCGCCTGGTCTGCTGCGGACGCCACTCCTGCGGCAGAGACCCGAGTGGCTCCCAGCAGTCGGCGAGCGCGGCTGAGGCTTCCGGGGGCCTGCGCCGCTGCCTGCCCCCGGGCCTTGATGGGAGCTTCAGCGGCTCGGAGCGCTCATCGCCCCAGCGCGACGGGCTGGACACCAGCGGCTCCACAGGCAGCCCCGGT The following amino acid sequence <SEQID NO. 26> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 25:NPIIYTLTNRDLRHALLRLVCCGRHSCGRDPSGSQQSASAAEASGGLRRCLPPGLDGSFSGSERSSPQRDGLDTSGSTGSPG The following DNA sequence beGPCR-seq21 <SEQ ID NO. 27> wasidentified in H. sapiens:CGTGAAGAACAGCGCCACCATGACCAGCATGTGCACCACGCGCGCTCTGCGCCGCGATGCTCGCGGGTCCGCAGCCTCCTNNNNNNNNNNNNNNNNNNNNNNNNNNTGGCAGAGCTTGCGCGCGATGCGGGCGTACATGACCACGATGAGCGCCAGCGGCGCCAGGTAGATGTGCGAGAAGAGCACAGTGGTGTAGACCCTGCGCATGCCCTTCTCGGGCCAGGCCTCCCAGCAGGAGTAGAGAGGGTAGGAGCGGTTGCGGGCGTCCACCATGAAGTGGTGCTCCTCACGGGTGACGGTCAGCGTGACGGCCGAGGGACACATGATGAGCAGCGCCAGGGCCCAGATGACGGCGATGGTGACGAGCGCCTTCCGCAGGGTCAGCTTCTCGCGGAAAGGGTGCACGATGCAGCGGAACCT Thefollowing amino acid sequence <SEQ ID NO. 28> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 27:FRCIVHPFREKLTLRKALVTIAVIWALALLIMCPSAVTLTVTREEHHFMVDARNRSYPLYSCWEAWPEKGMRRVYTTVLFSHIYLAPLALIVVMYARIARKLCXXXXXXXXXXEAADPRASRRRARVVHMLVMVALFFT Thefollowing DNA sequence beGPCR-seq22 <SEQ ID NO. 29> was identified in H.sapiens:GCAGGGGGCGTGAGTCCTCAGGCACTTCTTGAGGTCCTTGTTGAGCAGGAAGCAGACAATTGGGTTGACGGCAGCCTGGGCGAAGCTCATCCAAACAGCATGGCCAGGTAGCGGTGGGGCACAGCACAGGCTTTCACAAACACTCGCCAGTAGCAGGCCACGATGTAGGGTGACCAGAGGAGCAGAAAGAGCAGTGTGATCGCGTAGAACATGCGGCCCAGCTGCTTTTCACCCTTGACCTCGTCCATGCCCAGTAGCCGCCGGCTGGCTGCATGCCCATTCTGCCGGATACCCAGCAGGGTTGGTGGCATGGGCCC The following amino acid sequence <SEQID NO. 30> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 29:GPMPPTLLGIRQNGHASRRLLGMDEVKGEKQLGRMFYAITLLFLLLWSPYIVACYWRVFVKACAVPHRYLATAVWMSFAQAAVNPIVCFLLNKDLKKCLRTHAPC The following DNA sequencebeGPCR-seq24 <SEQ ID NO. 31> was identified in H. sapiens:TATTCTGTAATGAAGAATGTCATTCACACTGCCATTGGCACATCCAGTGGCCTCACCTAGCATTGTGAAAGCCCTTCGGTTGGTGTATTGCCACTTCATTTTAAAAGGATGCACAAGTCCCTGGTGCCTTTCCACAGCAATGCAGGTCATAGTGAGGATTTCTGTCACAACAGCGGTAGACTGGACAAATGGCACCATCTTGCAAATGAAAGCACCTGCAGTAAGGAAATAGGATAAATCATACATCAAAACAAAAAGAATAAAGGTTTCATCTGTGTCTTTGTAATTATCACTATCAGTCCATTCTGAGCCTCTGCCAAAAAGTTTGATAATTGTAATTACTCTGTAGACACAThe following amino acid sequence <SEQ ID NO. 32> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 31:VYRVITIIKLFGRGSEWTDSDNYKDTDETFILFVLMYDLSYFLTAGAFICKMVPFVQSTAVVTEILTMTCIAVERHQGLVHPFKMKWQYTNRRAFTMLGEATGCANGSVNDILHYRI The following DNA sequencebeGPCR-seq27 <SEQ ID NO. 33> was identified in H. sapiens:GAGCAACATGATCTTTTTGAAGTACTTGACGGTGTCGTTCTTGACGGTCACGAAGCACAGAGTGTTGATCATGCTGTTGCTCATGGCGATGCACTCGACGATGTAGAAGGCAGTGAGGTAGTGCTTCTCCTTCACAAACACGGTGGGGAAGAAGTCGCGCACGATGGTGAAGCCGTAGAAGGGCGCCCAGCATAGCACGTAGGCGGTGAGGATGCACATGAGCACCAGGACCGTCTTCCTGCGGCAGCGCAGCCTCTTGCGGATCTGCTCTGTCTGGAATCCAGGGACCGCCTTGAACCAGAGCTCCCGGGAGATCCTGGCATAGCACAGGGTCATGGTGACCACGGGGCCCACGAATTCTATGCCAAAGATAAAGAGGAAGTAGGACTTGTAGTAGAGCTGCTGGTCCACAGGCCAGATCTGGCCGCAGAAGATCTTTTCCTGGCTCTTGACAATGACGAGGACCGTCTCGGTGGTGAAGTAGGCGGAAGGGATGGCGATCAGGATGGACACCGTCCACACCAAGGCAATCAGGCCAGTGGCTGTTTGGCACTTCATTCGTGGTCTCAGCGGATGGACAATAGCCAGATACCTAGGGCAAGAACACAAGTGGAGGCAGCC The followingamino acid sequence <SEQ ID NO. 34> is the predicted amino acid sequencederived from the DNA sequence of SEQ ID NO. 33:GCLHLCSCPRYLAIVHPLRPRMKCQTATGLIALVWTVSILIAIPSAYFTTETVLVIVKSQEKIFCGQIWPVDQQLYYKSYFLFIFGIEFVGPVVTMTLCYARISRELWFKAVPGFQTEQIRKRLRCRRKTVLVLMCILTAYVLCWAPFYGFTIVRDFFPTVFVKEKHYLTAFYIVECIAMSNSMINTLCFVTVKNDTVKYFKKIMLL Thefollowing DNA sequence beGPCR-seq28 <SEQ ID NO. 35> was identified in H.sapiens:CAGCCACACTGCAGTGATGAAATCAAATGTCCAACACCAACCATAGTCACCATTACTAACTAAGAAGCCACAAAACTTCCCTTCCAGGGTGTTCAGCAGCAGGGACAGGGCCCAGGGCAGGGCACACATGACAGTTGACAGGTTTCTTGGGCAGCAGCAGCAGTACCAGATAGGCCGCAGGACAGACAGGCAGCACTCAGTACTGATGGCACTCAGCATGCTCAGGCCTACAAGGTAGGCAAAGGTCATCACGCTGGTGAAGAAGCTAGGGAAATTGATGGAGATGGAACAGAAGAAGTTACTGAGGTACACCAGGCAATTTATAATCTGGAAGCAGAGGAAGAGGAAGTCGGCCCCGGCCAGGCTGAGGACGTAGACAGAGAAGGCGTTCCTGCGCATGCGGAAGCCCAGGAGCCAGAGCACAAACCCGTTTCCTACCAGCCCGACCAGGGCAATGAAAAGGATCAGGAAGACCGGGATCAG The followingamino acid sequence <SEQ ID NO. 36> is the predicted amino acid sequencederived from the DNA sequence of SEQ ID NO. 35:LIPVFLILFIALVGLVGNGFVLWLLGFRMRRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPSFFTSVMTFAYLVGLSMLSAISTECCLSVLRPIWYCCCCPRNLSTVMCALPWALSLLLNTLEGKFCGFLVSNGDYGWCWTFDFITAVWL The following DNA sequence beGPCR-seq31 <SEQ ID NO. 37>was identified in H. sapiens:GAGAGTCTGATTCTGACTTACATCACATATGTAGGCCTGGGCATTTCTATTTGCAGCCTGATCCTTTGCTTGTCCGTTGAGGTCCTAGTCTGGAGCCAAGTGACAAAGACAGAGATCACCTATTTACGCCATGTGTGCATTGTTAACATTGCAGCCACTTTGCTGATGGCAGATGTGTGGTTCATTGTGGCTTCCTTTCTTAGTGGCCCAATAACACACCACAAGGGATGTGTGGCAGCCACATTTTTTGGTCATTTCTTTTACCTTTCTGTATTTTTCTGGATGCTTGCCAAGGCACTCCTTATCCTCTATGGAATCATGATTGTTTTC The following amino acid sequence<SEQ ID NO. 38> is the predicted amino acid sequence derived from theDNA sequence of SEQ ID NO. 37:ESLILTYITYVGLGISICSLILCLSVEVLVWSQVTKTEITYLRHVCIVNIAATLLMADVWFIVASFLSGPITHHKGCVAATFFGHFFYLSVFFWMLAKALLILYGIMIVF The following DNA sequencebeGPCR-seq32 <SEQ ID NO. 39> was identified in H. sapiens:TTGTGTGGCAGTAGAGAGATGTCAGGCTTCAGAGTCAACAAGAACTGGATTTCAAACTGGATTTGAGGACCCCCACCTTTGGTAAGTGACTTATTATCTGCGAGCCTCTGTTTCTCTCTTCTTTAAATGAGGACAGTAAATCCCATACGGCAGGGTGGTGGGGAGAATCAGAGATGATACAGCTGGTGATCACATCTGGTTTGTGTTCCCAGGGGCACCAGACTAGGGTTTCTGAGCATGGATCCAACCGTCCCAGTCTTCGGTACAAAACTGACACCAATCAACGGACGTGAGGAGACTCCTTGCTACAATCAGACCCTGAGCTTCACGGTGCTGACGTGCATCATTTCCCTTGTCGGACTGACAGGAAACGCGGTAGTGCTCTGGCTCCTGGGCTACCGCATGCGCAGGAACGCTGTCTCCATCTACATCCTCAACCTGGCCGCAGCAGACTTCCTCTTCCTCAGCTTCCAGATTATACGTTCGCCATTACGCCTCATCAATATCAGCCATCTCATCCGCAAAATCCTCGTTTCTGTGATGACCTTTCCCTACTTTACAGGCCTGAGTATGCTGAGCGCCATCAGCACCGAGCGCTGCCTGTCTGTTCTGTGGCCCATCTGGTACC The following amino acidsequence <SEQ ID NO. 40> is the predicted amino acid sequence derivedfrom the DNA sequence of SEQ ID NO. 39:LCGSREMSGFRVNKNWISNWIGPPPLVSDLLSASLCFSLLMRTVNPIRQGGGENQRYSWSHLVCVPRGTRLGFLSMDPTVPVFGTKLTPINGREETPCYNQTLSFTVLTCIISLVGLTGNAVVLWLLGYRMRRNAVSIYILNLAAADFLFLSFQIIRSPLRLINISHLIRKILVSVMTFPYFTGLSMLSAISTERCLSVLWPIWY Thefollowing DNA sequence beGPCR-seq33 <SEQ ID NO. 41> was identified in H.sapiens:ACAGAAAGCAAGGCCACCAGGACCTTAGGCATAGTCATGGGAGTGTTTGTGTTGTGCTGGCTGCCCTTCTTTGTCTTGACGATCACAGATCCTTTCATTAATTTTACAACCCTTGAAGATCTGTACAATGTCTTCCTCTGGCTAGGCTATTTCAACTCTGCTTTCAATCCCATTTTATATGGCATGCTTTATCCTTGGTTTCGCAAGGCATTGAGGATGATTGTCACAGGCATGATCTTCCACCCTGACTCTTCCACCCTAAGCCTGTTTTCTGCCCATGCTTAGGCTGTGTTCATCATTCAATAGGACTCTTTTCTGG The following amino acid sequence <SEQ IDNO. 42> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 41:TESKATRTLGIVMGVFVLCWLPFFVLTITDPFINFTTLEDLYNVFLWLGYFNSAFNPILYGMLYPWFRKALRMIVTGMIFHPDSSTLSLFSAHAAVFIIQDSF The following DNA sequence beGPCR-seq34<SEQ ID NO. 43> was identified in H. sapiens:TAGGAATCTCAGAGAAGAAAGTAAGGAACCAGAAAACCATAAAAGAATGTAAATGGAAAAGAATCAGCAAATCTTATTCACTTATCACTAAATCTAAAATATGTCAAAATACATGAAGACAACAAATGCTTTAGAACAACTGTTGAATGTATTGTCCTACAACTTGGCATATGATCATGCTTGCCTCTCTATGTCCAAGTGTTTATTTTTGCAGTTGACCTTAATTTCAAGTTAGTTTTGAGGTCTCTACAGTAATGTTTTTAATCTGTCTCTACTTCTTCAGAAAATAAATTAGTTGTTGACGAATCAGTCCTTAAGACCTTGCCGCTTACAATAAGTTTTATTGCCTTCCCAAACCATTGGTAAAAGAAAGCATAAATCAAGGGGTTCATAGCTGAATTATAATAAACACACCAAACTAAAATCTCATAAACATAAGGAGGAGTTATAAAATTCATATAAGCATCAATCACTGCATCAACGAGGTATGGTAGCCAAGAGACAAGAAATGCTGC The following amino acid sequence <SEQ ID NO. 44> is the predictedamino acid sequence derived from the DNA sequence of SEQ ID NO. 43:LHQRGMVAKRQEMLAAFLVSWLPYLVDAVIDAYMNFITPPYVYEILVWCVYYNSAMNPLIYAFFYQWFGKAIKLIVSGKVLRTDSSTTNLFSEEVETDKHYCRDLKTNLKLRSTAKINTWTRGKHDHMPSCRTIHSTVVLKHLLSSCI The following DNA sequence beGPCR-seq35 <SEQ ID NO. 45> wasidentified in H. sapiens:CTGGAAAGAGGTCCTCGATCTATCCTCTACGCCGTCCTTGGTTTTGGGGCTGTGCTGGCAGCGTTTGGAAACTTACTGGTCATGATTGCTATCCTTCACTTCTAACAACTGCACACACCTACAAACTTTCTGATTGCGTCGCTGGCCTGTGCTGACTTCTTGGTGGGAGTCACTGTGATGCCCTTCAGCACAGTGAGGTCTGTGGAGAGCTGTTGGTACTTTGGGGACAGTTACTGTAAATTCCATACATGTTTTGACACATCTTTCTGTTTTGCTTCTTTATTTCATTTATGCTGTATCTCTGTTGATAGATACATTGCTGTTACTGATCCTCTGACCTATCCAACCAAGTTTACTGTGTCAGTTTCAGGGATATGCATTGTTCTTTCCTGGTTCTTTTCTGTCACATACAGCTTTTCGATCTTTTACACGGGAGCCAACGAAGAAGGAATTGAGGAATTAGTAGTTGCTCTAACCTGTGTAGGAGGCTGCCAGGCTCCACTGAATCAAAACTGGGTCCTACTTTGTTTTCTTCTATTCTTTATACCCAATGTCGCCATGGTGTTTATATACAGTAAGATATTTTTGGTGGCCAAGCATCAGGCTAGGAAGATAGAAAGTACAGCCAGCCAAGCTCAGTCCTTCTCAGAGAGTTACAAGGAAAGAGTAGCAAAAAGAGAGAGAAAGGCTGCCAAAACCTTGGGAATTGCTATGGCAGCATTTCTTThe following amino acid sequence <SEQ ID NO. 46> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 45:LERGPRSILYAVLGFGAVLAAFGNLLVMIAILHFQLHTPTNFLIASLACADFLVGVTVMPFSTVRSVESCWYFGDSYCKFHTCFDTSFCFASLFHLCCISVDRYIAVTDPLTYPTKFTVSVSGICIVLSWFFSVTYSFSIFYTGANEEGIEELVVALTCVGGCQAPLNQNWVLLCFLLFFIPNVAMVFIYSKIFLVAKHQARKIESTASQAQSFSESYKERVAKRERKAAKTLGIAMAAFL The following DNA sequence beGPCR-seq36 <SEQ IDNO. 47> was identified in H. sapiens:AACCAGGTGGCCTTACTCCTAAGACCCCTGGCCTTGTCTATGGCCTTTATCAACAGCTGTCTCAATCCAGTTCTCTATGTCTTCATTGGGCATGACTTCTGGGAGCACTTGCTCCACTCCCTGCTAGCTGCCTTAGAACGGGCACTTAGCGAGGAGCCAGATAGTGCCTGAATCCCAGCTCCCAGGCAGATGAGTCCTTTATAACATGACCCAATTTCCTACTCCATTTTCCCACCACTCAATCCTCTTCCCAAACAGCTCTACCATAATCCAACATCCAACAGAATTTAAGAGAATAAACCACAACTTTTAAGTGAGCTCTATGTGCTAGGTCATGTTTTAGAATACAACCTTAAGTGCCTGGAAGATGGAGGCAAGAAACAAACAAGGTCTCATTCTTTAGAGGAAGACAGTTCACCAAGACTCAAACAGAAAAAAAGATAGTTATCTTGTGACAAAACAAGTCATAAAATTGGGTCAGGACCTGCAGCAATGACTTTATGCTAGAATCCAGAGCACTAGCAGGAAACTGCTTAAATTTTACTTAATCAAAGTCAAGTTTGGACATACATGTCAGGTAAAACCTAGCAGAGATGAGCTACCTTGATTTTAAAACTTCAAGGGATAGCTCAATGTCATCAAGATCCTTTTGATGACTTG The following amino acid sequence <SEQ ID NO. 48> is the predictedamino acid sequence derived from the DNA sequence of SEQ ID NO. 47:NQVALLLRPLALSMAFINSCLNPVLYVFIGHDFWEHLLHSLLAALERALSEEPDSAIPAPRQMSPLHDPISYSIFPPLNPLPKQLYHNPTSNRIENKPQLLSELYVLGHVLEYNLKCLEDGGKKQTRSHSLEEDSSPRLKQKKRLSCDKTSHKIGSGPAAMTLCNPEHQETAILLNQSQVWTYMSGKTQRATLILKLQGIAQCHQDPFDDL Thefollowing DNA sequence beGPCR-seq37 <SEQ ID NO. 49> was identified in H.sapiens:GCTTGTTCACGGCCACCATCCTCAAGCTGTTGCGCACGGAGGAGGCGCACGGCCGGGAGCAGCGGAGGCGCGCGGTGGGCCTGGCCGCGGTGGTCTTGCTGGCCTTTGTCACCTGCTTCGCCCCCAACAACTTCGTGCTCCTGGCGCACATCGTGAGCCGCCTGTTCTACGGCAAGAGCTACTACCACGTGTACAAGCTCACGCTGTGTCTCAGCTGCCTCAACAACTGTCTGGACCCGTTTGTTTATTACTTTGCGTCCCGGGAATTCCAGCTGCGCCTGCGGGAATATTTGGGCTGCCGCCGGGTGCCCAGAGACACCCTGGACACGCGCCGCGAGAGCCTCTTCTCCGCCAGGACCACGTCCGTGCGCTCCGAGGCCGGTGCGCACCCTGAAGGGATGGAGGGAGCCACCAGGCCCGGCCTCCAGAGGCAGGAGAGTGTGTTCTGAGTCCCGGGGGCGCAGC The following amino acid sequence <SEQ IDNO. 50> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 49:LFTATILKLLRTEEAHGREQRRRAVGLAAVVLLAFVTCFAPNNFVLLAHIVSRLFYGKSYYHVYKLTLCLSCLNNCLDPFVYYFASREFQLRLREYLGCRRVPRDTLDTRRESLFSARTTSVRSEAGAHPEGMEGATRPGLQRQESVFVPGAQAAPPGLR The following DNA sequence beGPCR-seq38 <SEQ ID NO. 51>was identified in H. sapiens:TTACTTATTCTGCCCTTTATCCAACTTTTAATTCCCTTTGCTATTCTCCTGCCTCATTTTCTGGCCTCATTTTCCCTATTATCCTGCCTCACATTGATCAAGGGATGAGGCTGGCAGGATCCGGAACCCACAGGGCCCCGTGGGCCATGAGAGGCTCCTGGACTTGAACCTCAGGACACTCCCACTCTGGCTGCCGGCAGGGATGGAAGCTGGATGAGCAGGCAGGAGCTGGCAGTGGGGGTGGAGAGCCATAGGCTATTGGGGTGGACAGGCTTGGGTGCCTCATGGGAGCTCCCCATGGGAGCTGTGGCCCCTTGGGGCCTCTTATTTCTCACCCCAGGCTTTCCCGGGAGAGGTTCAAGTCAGAAGATGCCCCAAAGATCCACGTGGCCCTGGGTGGCAGCCTGTTCCTCCTGAATCTGGCCTTCTTGGTCAATGTGGGGAGTGGCTCAAAGGGGTCTGATGCTGCCTGCTGGGCCCGGGGGGCTGTCTTCCACTACTTCCTGCTCTGTGCCTTCACCTGGATGGGCCTTGAAGCCTTCCACCTCTACCTGCTCGCTGTCAGGGTCTTCAACACCTACTTCGGGCACTACTTCCTGAAGC The following amino acid sequence <SEQ ID NO. 52> isthe predicted amino acid sequence derived from the DNA sequence of SEQID NO. 51:ETYSALYPTFNSLCYSPASFSGLIFPIILPHIDQGMRLAGSGTHRAPWAMRGSWTTSGHSHSGCRQGWKLDEQAGAGSGGGEPAIGVDRLGCLMGAPHGSCGPLGPLISHPRLSRERFKSEDAPKIHVALGGSLFLLNLAFLVNVGSGSKGSDAACWARGAVFHYFLLCAFTWMGLEAFHLYLLAVRVFNTYFGHYFL The following DNAsequence beGPCR-seq40 <SEQ ID NO. 53> was identified in H. sapiens:AATTGGTCGGAGAGTGCAGCTGCTTGAAATGGAGGATTGAAATCATCACCAGGAGGTTTCCAAACACAGCCAGCACAGCCCCAAAGCCAAACACTATGTACAGAATCACCCGGGATCCCGGCGAGAAGGGGATTTTCACACAGGACCCATTCACGTTCGCGTAGCACAGCTGCACAGCCACCAGCAGGGATGAATTGCTGCTCATAACGCTGGTATTTACATATGGAGAAATTTTGTCCTTGTTGATTATCACAAAAAATACAGGATTGTTCCTGATTTTCATTGCTCCTGCGGAAAAAAACACATATTCACCAGGATGCCAGAGGAAATGATCA The following amino acidsequence <SEQ ID NO. 54> is the predicted amino acid sequence derivedfrom the DNA sequence of SEQ ID NO. 53:DHFLWHPGEYVFFSAGAMKIRNNPVFFVIINKDKISPYVNTSVMSSNSSLLVAVQLCYANVNGSCVKIPFSPGSRVILYIVFGFGAVLAVFGNLLVMISILHFKQLHSPTN The following DNA sequencebeGPCR-seq41 <SEQ ID NO. 55> was identified in H. sapiens:CACATCTTAACAAGACTGAAAAACATTGATTTGTTTTTAATTTGAAGAGCAATTTATTTGCTATTCATTCATAGTCTTACTTGATTTTTAAAAACTCATTTCGCTTGGTAATTTTAAAGGTATCCTGAACTTCGTCTATCCAACTGCTTATATATGTTCAGAAAACAAATTCATGGTTGCTGAACTGTTCTTTAAAACCTGACCAGTTACAATAACTTTTATTGCTTTCCTAAACCATGGGTAAAATAAAGCATAAATCAAAGGATTCATGGCTGAGTTATAATAAGCACACCAACAGCATCATAAATACAGGCAGGGGTTATAAAGCCCATAAAGGCATCAATTAATGAATCAATGCTATATGGTAACCATGAAATCATAAATGCTACCACTGTGACCCCCAGGGTTTTAGCTGCTTTTCTCTCTCTCCTGGCCACTCTGGCTTTGTAACTCTCTGAGGATGATTCTGTCTTGCTACCAGTATTTTCTATCTTTTTCGCCTGTCGTCTAGCCACAAGAAATATGTTACCATACAGAATTATCATAATAAAGGTAGGTATAAAGAAGGATAGAAAATCTGTCAACA The following amino acid sequence <SEQ ID NO. 56> is the predictedamino acid sequence derived from the DNA sequence of SEQ ID NO. 55:LTDFLSFFIPTFIMIILYGNIFLVARRQAKKIENTGSKTESSSESYKARVARRERKAAKTLGVTVVAFMISWLPYSIDSLIDAFMGFITPACIYEICCWCAYYNSAMNPLIYALFYPWFRKAIKVIVTGQVLKNSSATMNLFSEHIAVGTKFRIPLKLPSEMSFKSSKTMNEQINCSSNKQINVFQSCDV The following DNA sequencenGPCR-seq53 <SEQ ID NO. 57> was identified in H. sapiens:TTTGTGGCAAGGAGACCCTGATCCCGGTCTTCCTGATCCTTTTCATTGCCCTGGTCGGGCTGGTAGGAAACGGGTTTGTGCTCTGGCTCCTGGGCTTCCGCATGCGCAGGAACGCCTTCTCTGTCTACGTCCTCAGCCTGGCCGGGGCCGACTTCCTCTTCCTCTGCTTCCAGATTATAAATTGCCTGGTGTACCTCAGTAACTTCTTCTGTTCCATCTCCATCAATTTCCCTAGCTTCTTCACCACTGTGATGACCTGTGCCTACCTTGCAGGCCTGAGCATGCTGAGCACCGTCAGCACCGAGCGCTGCCTGTCCGTCCTGTGGCCCATCTGGTATCGCTGCCGCCGCCCCAGACACCTGTCAGCGGTCGTGTGTGTCCTGCTCTGGGCCCTGTCCCTACTGCTGAGCATCTTGGAAGGGAAGTTCTGTGGCTTCTTATTTAGTGATGGTGACTCTGGTTGGTGTCAGACATTTGATTTCATCACTGCAGCGTGGCTGATTTTTTTATTCATGGTTCTCTGTGGGTCCAGTCTGGCCCTGCTGGTCAGGATCCTCTGTGGCTCCAGGGGTCTGCCACTGACCAGGCTGTACCTGACCATCCTGCTCACAGTGCTGGTGTCCCTCCTCTGCGGCCTGCCCTTTGGCATTCAGTGGTTCCTAATATTATGGATCTGGAAGGATTCTGATGTCTTATTTTGTCATATTCATCCAGTTTCAGTTGTCCTGTCATCTCTTAACAGCAGTGCCAACCCCATCATTTACTTCTTCGTGGGCTCTTTTAGGAAGCAGTGGCGGSTGCAGCACCCGATCCTCAAGCTGGCTCTCCAGAGGGCTCTGCAGGACATTGCTGAGGTGGATCACAGTGAAGGATGCTTCCGTCAGGGCACCCGGAGATTCAAAGAAGCATTCTGGTGTAGGGATGGACCCCTCTACTTCCATCATATATATGTGGCTTTGAGAGGCAACTTTGCCCC The following amino acid sequence <SEQ IDNO. 58> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 57:CGKETLIPVFLILFIALVGLVGNGFVLWLLGFRMRRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPSFFTTVMTCAYLAGLSMLSTVSTERCLSVLWPIWYRCRRPRHLSAVVCVLLWALSLLLSILEGKFCGFLFSDGDSGWCQTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVSLLCGLPFGIQWFLILWIWKDSDVLFCHIHPVSVVLSSLNSSANPIIYFFVGSFRKQWRXQHPILKLALQRALQDIAEVDHSEGCFRQGTRRFKEAFWCRDGPLYFHHIYVALRGNFA The following DNA sequence nGPCR-seq54<SEQ ID NO. 59> was identified in H. sapiens:CTTTGCATCTCACTGTTGAGCAGACAGCCTGCTGAAAGTTGTCGCTGACCACCACATATAGTAACAGGTTACCAAAGGTGTTCAGAGCAGCATAATGGTCTAGAAACGATGTAAGCTTCATGGATCTGATTCTCAATGGAACAACTGATTGAAAGCAGGCTGAGATTCGATCCTGAATGACCCTCAAGATATGGAAGGGTAAAAAACATACGTAAAATGCAAGGAGTAGCAGAATGGTTAGCCTTCGTGCTTTCTGCTTAAGGCAGCTGTCAGTTTGCAGTCCATGGGTCAAAGTGTGGATAATCGTGGTATAGCAAAGTGTCACTATCACCAAGGGGAGGCAGAAAGTACTTGCAGTCAAAATCAGGTTGTACCACTTAATAGTATTGAGTTCATCCGAACTGGTGAGGTCGAGACAGGCTGATCTGTTGGTCCTGTTGGTTGATGTGATCAAGAAGGTCATCGGAATGACAGCTACCAGTGAAATGATCCACACCACAGCACAGGCTACAACTGCACATCGAGTTTTGTGAATGGAAAAGCAGCTCATTGGGTGAATGATCACACAGTAGCGGAAG Thefollowing amino acid sequence <SEQ ID NO. 60> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 59:FRYCVIIHPMSCFSIHKTRCAVVACAVVWIISLVAVIPMTFLITSTNRTNRSACLDLTSSDELNTIKWYNLILTASTFCLPLVIVTLCYTTIIHTLTHGLQTDSCLKQKARRLTILLLLAFYVCFLPFHILRVIQDRISACFQSVVPLRIRSMKLTSFLDHYAALNTFGNLLLYVVVSDNFQQAVCSTVRCK The following DNAsequence nGPCR-seq55 <SEQ ID NO. 61> was identified in H. sapiens, wherethe underlined ATG identifies a probable start codon:GGGAGGGCTCGTAGACACACTAACCCTACCCTTTCTGTTTCTTCCTCATCTTTCCTTTCCATCTGTTTCTCATGGTCTCCTGTCTGTCTCTCTCTCTCTCCCCTCTTTCTCTCTCCTCGCTCTTTCTCATCCCCTCCATTTCTGTGTCAATCTCAATCCATTTATATCGGTGGCCACTTTTCTATCTCTTTGTTCTATCTCTCTCTCTCTCTCTTTCCCACTTTGTCTCTGCACGCCTGTTGTGTTTTTCTGCCTGTCTCTCTCTTGCCCTCATCTCTCTGTCTCTCTCTTGCCCTCATCTCTCTGTCTCTCTGTGTCTGTGTCTCCCCCGCTCATTCCCATTTGCAGGTGCAATGTAGCAGGACAACTCATGGAGCCCCCCCGGGCCCATCGAGTACCGGACTGGCTGACCCCCTAGGGTTGGCAGTAGCCCCTGACCCTCAGTATGGCCAACACTACCGGAGAGCCTGAGGAGGTGAGCGGCGCTCTGTCCCCACCGTCCGCATCAGCTTATGTGAAGCTGGTACTGCTGGGACTGATTATGTGCGTGAGCCTGGCGGGTAACGCCATCTTGTCCCTGCTGGTGCTCAAGGAGCGGGCCCTGCACAAGGCTCCTTACTACTTCCTGCTGGACCTGTGCCTGGCCGATGGCATACGCTCTGCCGTCTGCTTCCCCTTTGTGCTGGCTTCTGTGCGCCACGGCTCTTCATGGACCTTCAGTGCACTCAGCTGCAAGATTGTGGCCTTTATGGCCGTGCTCTTTTGCTTCCATGCGGCCTTCATGCTGTTCTGCATCAGCGTCACCCGCTACATGGCCATCGCCCACCACCGCTTCTACGCCAAGCGCATGACACTCTGGACATGCGCGGCTGThe following amino acid sequence <SEQ ID NO. 62> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 61:MANTTGEPEEVSGALSPPSASAYVKLVLLGLIMCVSLAGNAILSLLVLKERALHKAPYYFLLDLCLADGIRSAVCFPFVLASVRHGSSWTFSALSCKIVAFMAVLFCFHAAFMLFCISVTRYMAIAHHRFYAKRMTLWTCAAEThe following DNA sequence nGPCR-seq56 <SEQ ID NO. 63> was identified inH. sapiens:AAAAATTGCTGTACTGAACTATTGAATGGAACTTGGAAATAAAGTCCCTTCCAAAATAACTATTCTTCAACAGAGAGTAATAGGTAAATGTTTTAGAAGTGAGAGGACTCAAATTGCCAATGATTTACTCTTTTATTTTTCCTCCTAGGTTTCTGGGATAAGTATGTGCAAATAAAAAATAAACATGAGAAGGAACTGTAACCTGATTATGGATTTGGGAAAAAGATAAATCAACACACAAAGGGAAAAGTAAACTGATTGACAGCCCTCAGGAATGATGCCCTTTTGCCACAATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTGCTTCCCTGTACAGTTTAATGGTGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTATATCACACTTCAAACAACTTCATACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTGGGGTGTCTGGTCATGCCTTACAGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTAAAATTCACACAAGCACCGACATTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCGCTACTATGCTGTGTGTGATCCACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTCATTAGTTGGAGTGTCCCTGCTGTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAGAGATATATTACAAACATGTTCACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGACCTTTATGACTTCTTTTTATATACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAAGAACAGGCAAGATTAATTAGTGATGCCAATCAGA The following amino acid sequence <SEQ ID NO. 64> is thepredicted amino acid sequence derived from the DNA sequence of SEQ IDNO. 63:REKTDQPSGMMPFCRNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFLLGCLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNILVICVMIFISWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVYYRIYLIAKEQARLISDANQ The following DNA sequence nGPCR-seq57 <SEQ ID NO.65> was identified in H. sapiens:AACAGTCCCGGGTGGAACCTGGGCATGTATATTTTGATTGTTTTATGCATACTCCTAGTGAAGAACCAATGTCTTGCTCAGATAGAAGCAAGATACTCAGACTTAGTTTCTCTGTAGCTCCTGCTTTTTATTATTCCTGGTTGGATTGCACCACTACTCAGTTTCTATTTTATAATACTGATTATAAAACATGGGAGGGAAATAACTTTGTATTGGTTTTTATGGATAATTTATTATGTGTCCTAGACTCTGGCCTTGTCAAAAGAAGGACGTAAGAAGGCACGATGTATTATACTTGGGAATGATAGAAGAGACTGACCTGGTATTTCCACCCGGAAGAGGGAAAGGATTTTAACTACAAATACAGGAATCCAGCAGATGGCATCAGAGAACACTATAAAAAAGAAACGATTTGCAACAGCCACCTCTCTTCCAAAACAATTCCTTACTTCTGTGGTCTGCAAGGCGGTTTTTTGAATGGAACAGAACATAGTAATATAGGAAAACACAATGATGAGAAAAGCCAGCAAGTTCACACCTGTTGGGGAAAAGCACACTTTTAACATCTCAGGCGTAAAAGTCAACAGTAAAATTACTGTGGTACAGGTTGAGTATCCCTTACCCAAAATGTTTGAAACCAGAAATGTTTTGGATTTCGGATTTCGGAATATTTACACATTCATAATGATATATCTTGGAAATGGTTCCCAAGTCTAAACACAAAATTTATTTATGTTTCATATACACCTTATACACATAGTCTGAAAGTAATTTTGTACAATATTTTAAATAATTTTGGGCATGAAACAAAGTTTGCATACATTGAACCATCAGACAGCAAAAGCTTCAGGTGTGGAATTTTCCACTTGTGGCATCATGTTGATGCTCAAAAAGTTCCATATTTTAGAGCATTTCAAATTTTGGATTTTCAAATTACAAATGCTTAACCTGTACTTAGATGTTAAATACAGTGCCTCTTCCACGGGCACTTTCAGGAAGCATTCTTTTATATAAGCCCThe following amino acid sequence <SEQ ID NO. 66> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 65:YIKECFLKVPVEEALYLTSKYRLSICNLKIQNLKCSKIWNFLSINMMPQVENSTPEAFAVWFNVCKLCFMPKIINIVQNYFQTMCIRCININKFCVTWEPFPRYIIMNVIFRNPKSKTFLVSNILGKGYSTCTTVILLLTFTPEMLKVCFSPTGVNLLAFLIIVFSYITMFCSIQKTALQTTEVRNCFGREVAVANRFFFIVFSDAICWIPVFVVKILSLFRVEIPGQSLLSFPSIIHRAFLRPSFDKARVDTIIHKNQYKVISLPCFIISIIKKLSSGAIQPGIIKSRSYRETKSEYLASIARHWFFTRSMHKTIKIYMPRFHPGL The following DNA sequencenGPCR-seq58 <SEQ ID NO. 67> was identified in H. sapiens:ACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGATTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGCACCTGCTGCC The following amino acid sequence <SEQ ID NO. 68> is thepredicted amino acid sequence derived from the DNA sequence of SEQ IDNO. 67:TTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAA The following DNA sequence nGPCR-seq59 <SEQ ID NO. 69> wasidentified in H. sapiens:TACAGGCCTGAGCATGCTGGGCTCCATCAGCACCAAGCACTGCCTGTCCATCCTGTGGCCCATCTAGTACCGCTGCCACCACCCCACACACCTGTCAGCAGTCGTGTGTCCTGCTCTGGGCCCTGTCCCTGCTGCAGAGCATCCTGGAATGGATGTTCTGTGGCTTCCTGTCTAGTGGTGCTGATTCTGTTTGGTGTGAAACATCAGATTTCATCACAGTCACATGGCTGATTTTTTTATGTGTGGTTCTCTGCGGGTCCAGCCCGGTTCTGCTGGTCAGGATCCTTTGTGGATCCCGGAAGATGCCCTTGACCAGGCTGTACATGACCATCCTGCTCAGAGTGCTGGTCTTCCTCCTCTGTGACCTGCCCTTTGGCATTCAGTGATTCCTATTTTTCTGGATCCACGTGGATTTGTCACGTTCGTCTAGTTTCCATTTTCCTGTCCACTCTTAACAGCAGTGCCAACCCCATTATTTACTTCTTCATGGGCTCCTTTAGGCAGCTTCAAAACAGGAAGACTCTCTAGCTGGTTCTCCAGAGGGCTCTGCAGGACACGCCTGAGGTGGAAGAAGGCAGATGGCGGCTTTCTGAGGAAACCCTGGAGCTGTCATGAAGCAGATTGGGGCCATGAGGAAGAGCCTCTGCCCTGTCAGTCAG The following amino acid sequence <SEQ ID NO. 70> is the predictedamino acid sequence derived from the DNA sequence of SEQ ID NO. 69:YRPEHAGLHQHQALPVHPVAHLVPLPPPHTPVSSRVSCSGPCPCCRASWNGCSVASCLVVLILFGVKHQISSQSHGFFYVWFSAGPARFCWSGSFVDPGRCPPGCTPSCSECWSSSSVTCPLAFSDSYFSGSTWICHVRLVSIFLSTLNSSANPIIYFFMGSFRQLQNRKTLLVLQRALQDTPEVEEGRWRLSEETLELSSRLGPGRASALSV Thefollowing DNA sequence nGPCR-seq60 <SEQ ID NO. 71> was identified in H.sapiens:ATGCCGAAGGCAGGCCGCAGAAGAGAAGAGGAGGACGGTGAGGAGGATGAGCCCAGGGAAGCCCCGGGGTGGGGGCCGCTGGGGGCCTCGCTCCACCCGCAGCAGCAGCATAAGGCTGGCCCCACACATGGTGCAACACAGCAGAGCCAGCAGCACCGCTGCCACCAGCCACAGCGTCCGGCACAAGTGGCGGCTGGGCTCCCCGAAGAACTGGGTGCAGGCGCCGCTGAGCAGCAGGTGCAGCAGCAGGCAGAGGGCCCAGGTGAGGGCGCACACACAGGTGGTCAGGTGGCGTGGGCGGCGGCACGAGTACCAGGCTGGGAAGAGGGCGGCCAGGCACTGCTCCACGCTGACGGCCGCCAGGAGACTCAGGCCCACGATGTAGCAGAAGAAGCGCAGCGTTGCCAGGCTGGTCTGCACGAAGCCCGGGAAGTCCAGCCGGCCTTGCAGCAAGTCGGGGACGATGGCCACCATGTGGCAGCCAAGGAAGATGAGATCCGCGCAGGCCACGTCCAGGAGGTAGATGGCGAAAGGGTTTCTGTAGACATTGGAGCTGAGC The following aminoacid sequence <SEQ ID NO. 72> is the predicted amino acid sequencederived from the DNA sequence of SEQ ID NO. 71:LSSNVYRNPFAIYLLDVACADLIFLGCHMVAIVPDLLQGRLDFPGFVQTSLATLRFFCYIVGLSLLAAVSVEQCLAALFPAWYSCRRPRHLTTCVCALTWALCLLLHLTTCVCALTWALCLLLHLLLSGACTLLLSGACTQFFGEPSRHLCRTLWLVAAVLLALLCCTMCGASLMLLLRVERGPQRPPPRGFPGLILLTVLLFSSAACLRH The following DNA sequence nGPCR-1 <SEQ ID NO. 73> wasidentified in H. sapiens:ATGGAATCATCTTTCTCATTTGGAGTGATCCTTGCTGTCCTGGCCTCCCTCATCATTGCTACTAACACACTAGTGGCTGTGGCTGTGCTGCTGTTGATCCACAAGAATGATGGTGTCAGTCTCTGCTTCACCTTGAATCTGGCTGTGGCTGACACCTTGATTGGTGTGGCCATCTCTGGCCTACTCACAGACCAGCTCTCCAGCCCTTCTCGGCCCACACAGAAGACCCTGTGCAGCCTGCGGATGGCATTTGTCACTTCCTCCGCAGCTGCCTCTGTCCTCACGGTCATGCTGATCACCTTTGACAGGTACCTTGCCATCAAGCAGCCCTTCCGCTACTTGAAGATCATGAGTGGGTTCGTGGCCGGGGCCTGCATTGCCGGGCTGTGGTTAGTGTCTTACCTCATTGGCTTCCTCCCACTCGGAATCCCCATGTTCCAGCAGACTGCCTACAAAGGGCAGTGCAGCTTCTTTGCTGTATTTCACCCTCACTTCGTGCTGACCCTCTCCTGCGTTGGCTTCTTCCCAGCCATGCTCCTCTTTGTCTTCTTCTACTGCGACATGCTCAAGATTGCCTCCATGCACAGCCAGCAGATTCGAAAGATGGAACATGCAGGAGCCATGGCTGGAGGTTATCGATCCCCACGGACTCCCAGCGACTTCAAAGCTCTCCGTACTGTGTCTGTTCTCATTGGGAGCTTTGCTCTATCCTGGACCCCCTTCCTTATCACTGGCATTGTGCAGGTGGCCTGCCAGGAGTGTCACCTCTACCTAGTGCTGGAACGGTACCTGTGGCTGCTCGGCGTGGGCAACTCCCTGCTCAACCCACTCATCTATGCCTATTGGCAGAAGGAGGTGCGACTGCAGCTCTACCACATGGCCCTAGGAGTGAAGAAGGTGCTCACCTCATTCCTCCTCTTTCTCTCGGCCAGGAATTGTGGCCCAGAGAGGCCCAGGGAAAGTTCCTGTCACATCGTCACTATCTCCAGCTCAGAGTTTGATGGCTAA Thefollowing amino acid sequence <SEQ ID NO. 74> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 73:MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPTQKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLGIPMFQQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPSDFKALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLYHMALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG The following DNA sequenceTL-GPCR-seq5 <SEQ ID NO. 75> was identified in H. sapiens.AACTGGAAGGGCAGCCGTCTGCCGCCCACGAACACCTTCTCAAGCACTTTGAGTGACCACGGCTTGCAAGCTGGTGGCTGGCCCCCCGAGTCCCGGGCTCTGAGGCACGGCCGTCGACTTAAGCGTTGCATCCTGTTACCTGGAGACCCTCTGAGCTCTCACCTGCTACTTCTGCCGCTGCTTCTGCACAGAGCCCGGGCGAGGACCCCTCCAGGATGCAGGTCCCGAACAGCACCGGCCCGGACAACGCGACGCTGCAGATGCTGCGGAACCCGGCGATCGCGGTGGCCCTGCCCGTGGTGTACTCGCTGGTGGCGGCGGTCAGCATCCCGGGCAACCTCTTCTCTCTGTGGGTGCTGTGCCGGCGCATGGGGCCCAGATCCCCGTCGGTCATCTTCATGATCAACCTGAGCGTCACGGACCTGATGCTGGCCAGCGTGTTGCCTTTCCAAATCTACTACCATTGCAACCGCCACCACTGGGTATTCGGGGTGCTGCTTTGCAACGTGGTGACCGTGGCCTTTTACGCAAACATGTATTCCAGCATCCTCACCATGACCTGTATCAGCGTGGAGCGCTTCCTGGGGGTCCTGTACCCGCTCAGCTCCAAGCGCTGGCGCCGCCGTCGTTACGCGGTGGCCGCGTGTGCAGGGACCTGGCTGCTGCTCCTGACCGCCCTGTCCCCGCTGGCGCGCACCGATCTCACCTACCCGGTGCACGCCCTGGGCATCATCACCTGCTTCGACGTCCTCAAGTGGACGATGCTCCCCAGCGTGGCCATGTGGGCCGTGTTCCTCTTCACCATCTTCATCCTGCTGTTCCTCATCCCGTTCGTGATCACCGTGGCTTGTTACACGGCCACCATCCTCAAGCTGTTGCGCACGGAGGAGGCGCACGGCCGGGAGCAGCGGAGGCGCGCGGTGGGCCTGGCCGCGGTGGTCTTGCTGGCCTTTGTCACCTGCTTCGCCCCCAACAACTTCGTGCTCCTGGCGCACATCGTGAGCCGCCTGTTCTACGGCAAGAGCTACTACCACGTGTACAAGCTCACGCTGTGTCTCAGCTGCCTCAACAACTGTCTGGACCCGTTTGTTTATTACTTTGCGTCCCGGGAATTCCAGCTGCGCCTGCGGGAATATTTGGGCTGCCGCCGGGTGCCCAGAGACACCCTGGACACGCGCCGCGAGAGCCTCTTCTCCGCCAGGACCACGTCCGTGCGCTCCGAGGCCGGTGCGCACCCTGAAGGGATGGAGGGAGCCACCAGGCCCGGCCTCCAGAGGCAGGAGAGTGTGTTCTGAGTCCCGGGGGCGCAGCTTGGAGAGCCGGGGGCGCAGCTTGGAGGATCCAGGGGCGCATGGAGAGGCCACGGTGCCAGAGGTTCAGGGAGAACAGCTGCGTTGCTCCCAGGCACTGCAGAGGCCCGGTGGGGAAGGGTCTCCAGGCTTTATTCCTCCCAGGCACTGCAGAGGCACCGGTGAGGAAGGGTCTCCAGGCTTCACTCAGGGTAGAGAAACAAGCAAAGCCCAGCAGCGCACAGGGTGCTTGTTATCCTGCAGAGGGTGCCTCTGCCTCTCTGTGTCAGGGGACAGCTTGTGTCACCACGCCCGGCTAATTTTTGTATTTTTTTTAGTAGAGCTGGGCTGTCACCCCCGAGCTCCTTAGACACTCCTCACACCTGTCCATACCCGAGGATGGATATTCAACCAGCCCCACCGCCTACCCGACTCGGTTTCTGGATATCCTCTGTGGGCGAACTGCGAGCCCCATTCCCAGCTCTTCTCCCTGCTGACATCGTCCCTTAGCACACCTGTCCATACCCGAGGATGGATATTCAACCAGCCCCACCGCCTACCCGACTCGGTTTCTGGATATCCTCTGTGGGCGAACTGCGAGCCCCATTCCCAGCTCTTCTCCCTGCTGACATCGTCCCTTAGTTGTGGTTCTGGCCTTCTCCATTCTCCTCCAGGGGTTCTGGTCTCCGTAGCCCGGTGCACGCCGAAATTTCTGTTTATTTCACTCAGGGGCACTGTGGTTGCTGTGGTTGGAATTCTTCTTTCAGAGGAGCGCCTGGGGCTCCTGCAAGTCAGCTACTCTCCGTGCCCACTTCCCCTCACACACACACCCCCCTCGTGCCGAATTC The following amino acid sequence <SEQ ID NO. 76>is the predicted amino acid sequence derived from the DNA sequence ofSEQ ID NO. 75.MQVPNSTGPDNATLQMLRNPAIAVALPVVYSLVAAVSIPGNLFSLWVLCRRMGPRSPSVIFMINLSVTDLMLASVLPFQIYYHCNRHHWVFGVLLCNVVTVAFYANMYSSILTMTCISVERFLGVLYPLSSKRWRRRRYAVAACAGTWLLLLTALSPLARTDLTYPVHALGIITCFDVLKWTMLPSVAMWAVFLFTIFILLFLIPFVITVACYTATILKLLRTEEAHGREQRRRAVGLAAVVLLAFVTCFAPNNFVLLAHIVSRLFYGKSYYHVYKLTLCLSCLNNCLDPFVYYFASREFQLRLREYLGCRRVPRDTLDTRRESLFSARTTSVRSEAGAHPEGMEGATRPGLQRQESVF Thefollowing DNA sequence nGPCR-9 <SEQ ID NO. 77> was identified in H.sapiens:ATGGAGTCGGGGCTGCTGCGGCCGGCGCCGGTGAGCGAGGTCATCGTCCTGCATTACAACTACACCGGCAAGCTCCGCGGTGCGCGCTACCAGCCGGGTGCCGGCCTGCGCGCCGACGCCGTGGTGTGCCTGGCGGTGTGCGCCTTCATCGTGCTAGAGAATCTAGCCGTGTTGTTGGTGCTCGGACGCCACCCGCGCTTCCACGCTCCCATGTTCCTGCTCCTGGGCAGCCTCACGTTGTCGGATCTGCTGGCAGGCGCCGCCTACGCCGCCAACATCCTACTGTCGGGGCCGCTCACGCTGAAACTGTCCCCCGCGCTCTGGTTCGCACGGGAGGGAGGCGTCTTCGTGGCACTCACTGCGTCCGTGCTGAGCCTCCTGGCCATCGCGCTGGAGCGCAGCCTCACCATGGCGCGCAGGGGGCCCGCGCCCGTCTCCAGTCGGGGGCGCACGCTGGCGATGGCAGCCGCGGCCTGGGGCGTGTCGCTGCTCCTCGGGCTCCTGCCAGCGCTGGGCTGGAATTGCCTGGGTCGCCTGGACGCTTGCTCCACTGTCTTGCCGCTCTACGCCAAGGCCTACGTGCTCTTCTGCGTGCTCGCCTTCGTGGGCATCCTGGCCGCTATCTGTGCACTCTACGCGCGCATCTACTGCCAGGTACGCGCCAACGCGCGGCGCCTGCCGGCACGGCCCGGGACTGCGGGGACCACCTCGACCCGGGCGCGTCGCAAGCCGCGCTCGCTGGCCTTGCTGCGCACGCTCAGCGTGGTGCTCCTGGCCTTTGTGGCATGTTGGGGCCCCCTCTTCCTGCTGCTGTTGCTCGACGTGGCGTGCCCGGCGCGCACCTGTCCTGTACTCCTGCAGGCCGATCCCTTCCTGGGACTGGCCATGGCCAACTCACTTCTGAACCCCATCATCTACACGCTCACCAACCGCGACCTGCGCCACGCGCTCCTGCGCCTGGTCTGCTGCGGACGCCACTCCTGCGGCAGAGACCCGAGTGGCTCCCAGCAGTCGGCGAGCGCGGCTGAGGCTTCCGGGGGCCTGCGCCGCTGCCTGCCCCCGGGCCTTGATGGGAGCTTCAGCGGCTCGGAGCGCTCATCGCCCCAGCGCGACGGGCTGGACACCAGCGGCTCCACAGGCAGCCCCGGTGCACCCACAGCCGCCCGGACTCTGGTATCAGAACCGGCTGCAGACTGA The following amino acid sequence <SEQ IDNO. 78> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 77:MESGLLRPAPVSEVIVLHYNYTGKLRGARYQPGAGLRADAVVCLAVCAFIVLENLAVLLVLGRHPRFHAPMFLLLGSLTLSDLLAGAAYAANILLSGPLTLKLSPALWFAREGGVFVALTASVLSLLAIALERSLTMARRGPAPVSSRGRTLAMAAAAWGVSLLLGLLPALGWNCLGRLDACSTVLPLYAKAYVLFCVLAFVGILAAICALYARIYCQVRANARRLPARPGTAGTTSTRARRKPRSLALLRTLSVVLLAFVACWGPLFLLLLLDVACPARTCPVLLQADPFLGLAMANSLLNPIIYTLTNRDLRHALLRLVCCGRHSCGRDPSGSQQSASAAEASGGLRRCLPPGLDGSFSGSERSSPQRDGLDTSGSTGSPGAPTAARTLVSEPAAD The following DNA sequence nGPcR-11<SEQ ID NO. 79> was identified in H. sapiens:ATGTACAACGGGTCGTGCTGCCGCATCGAGGGGGACACCATCTCCCAGGTGATGCCGCCGCTGCTCATTGTGGCCTTTGTGCTGGGCGCACTAGGCAATGGGGTCGCCCTGTGTGGTTTCTGCTTCCACATGAAGACCTGGAAGCCCAGCACTGTTTACCTTTTCAATTTGGCCGTGGCTGATTTCCTCCTTATGATCTGCCTGCCTTTTCGGACAGACTATTACCTCAGACGTAGACACTGGGCTTTTGGGGACATTCCCTGCCGAGTGGGGCTCTTCACGTTGGCCATGAACAGGGCCGGGAGCATCGTGTTCCTTACGGTGGTGGCTGCGGACAGGTATTTCAAAGTGGTCCACCCCCACCACGCGGTGAACACTATCTCCACCCGGGTGGCGGCTGGCATCGTCTGCACCCTGTGGGCCCTGGTCATCCTGGGAACAGTGTATCTTTTGCTGGAGAACCATCTCTGCGTGCAAGAGACGGCCGTCTCCTGTGAGAGCTTCATCATGGAGTCGGCCAATGGCTGGCATGACATCATGTTCCAGCTGGAGTTCTTTATGCCCCTCGGCATCATCTTATTTTGCTCCTTCAAGATTGTTTGGAGCCTGAGGCGGAGGCAGCAGCTGGCCAGACAGGCTCGGATGAAGAAGGCGACCCGGTTCATCATGGTGGTGGCAATTGTGTTCATCACATGCTACCTGCCCAGCGTGTCTGCTAGACTCTATTTCCTCTGGACGGTGCCCTCGAGTGCCTGCGATCCCTCTGTCCATGGGGCCCTGCACATAACCCTCAGCTTCACCTACATGAACAGCATGCTGGATCCCCTGGTGTATTATTTTTCAAGCCCCTCCTTTCCCAAATTCTACAACAAGCTCAAAATCTGCAGTCTGAAACCCAAGCAGCCAGGACACTCAAAAACACAAAGGCCGGAAGAGATGCCAATTTCGAACCTCGGTCGCAGGAGTTGCATCAGTGTGGCAAATAGTTTCCAAAGCCAGTCTGATGGGCAATGGGATCCCCACATTGTTGAGTGGCACTGA The following amino acid sequence <SEQ ID NO. 80> isthe predicted amino acid sequence derived from the DNA sequence of SEQID NO. 79:MYNGSCCRIEGDTISQVMPPLLIVAFVLGALGNGVALCGFCFHMKTWKPSTVYLFNLAVADFLLMICLPFRTDYYLRRRHWAFGDIPCRVGLFTLAMNRAGSIVFLTVVAADRYFKVVHPHHAVNTISTRVAAGIVCTLWALVILGTVYLLLENHLCVQETAVSCESFIMESANGWHDIMFQLEFFMPLGIILFCSFKIVWSLRRRQQLARQARMKKATRFIMVVAIVFITCYLPSVSARLYFLWTVPSSACDPSVHGALHITLSFTYMNSMLDPLVYYFSSPSFPKFYNKLKICSLKPKQPGHSKTQRPEEMPISNLGRRSCISVANSFQSQSDGQWDPHIVEWH The following DNAsequence nGPCR-16 <SEQ ID NO. 81> was identified in H. sapiens:ATGACAGGTGACTTCCCAAGTATGCCTGGCCACAATACCTCCAGGAATTCCTCTTGCGATCCTATAGACACCCCACTTAATCAGCCTCTACTTCATAGTGCTTATTGGCGGGCTGGTGGGTGTCATTTCCATTCTTTTCCTCCTGGTGAAAATGAACACCCGGTCAGTGACCACCATGGCGGTCATTAACTTGGTGGTGGTCCACAGCGTTTTTCTGCTGACAGTGCCATTTCGCTTGACCTACCTCATCAAGAAGACTTGGATGTTTGGGCTGCCCTTCTGCAAATTTGTGAGTGCCATGCTGCACATCCACATGTACCTCACGTTCCTATTCTATGTGGTGATCCTGGTCACCAGATACCTCATCTTCTTCAAGTGCAAAGACAAAGTGGAATTCTACAGAAAACTGCATGCTGTGGCTGCCAGTGCTGGCATGTGGACGCTGGTGATTGTCATTGTGGTACCCCTGGTTGTCTCCCGGTATGGAATCCATGAGGAATACAATGAGGAGCACTGTTTTAAATTTCACAAAGAGCTTGCTTACACATATGTGAAAATCATCAACTATATGATAGTCATTTTTGTCATAGCCGTTGCTGTGATTCTGTTGGTCTTCCAGGTCTTCATCATTATGTTGATGGTGCAGAAGCTACGCCACTCTTTACTATCCCACCAGGAGTTCTGGGCTCAGCTGAAAAACCTATTTTTTATAGGGGTCATCCTTGTTTGTTTCCTTCCCTACCAGTTCTTTAGGATCTATTACTTGAATGTTGTGACGCATTCCAATGCCTGTAACAGCAAGGTTGCATTTTATAACGAAATCTTCTTGAGTGTAACAGCAATTAGCTGCTATGATTTGCTTCTCTTTGTCTTTGGGGGAAGCCATTGGTTTAAGCAAAAGATAATTGGCTTATGGAATTGTGTTTTGTGCCGTTAGCCACAAACTACAGTATTCATATTTGCTTCCTTTATATTGGGAATAAAAATGGGTATAGGGGAGGTAAGAATGGTATTTCATTACTTGATCAAAACCATGCCTTGATGTACCCAAAACAAAAGGACTATAAAATGCAAGAGCCCTCATTGTAGTCCTTATGGGATCCCTCCCATCTCTGAGTGATGGCCGTACAAAGACCAGTGTTGTTGAATCCACCTGGAGTTGCAATATTACATTATTTTCCAGTACAGAATGTCTGTGTGGCCCATGAAAGCAACATAGGTTTTAAGAGTTTTAGAGTTTCATTAGCTCATTCTAAGTTCCTCTGTTTGAAGCATGGTCTCTTAGGTTTTGGACTGAACTCAGACCTTTAGTTCTTTTCATCCCACTTCACCTTAGGTAAGTAAATTCTGGCCACCACCCAGCTCCAAAGACACAAACTCTCCTTCGCTAACCAGGTTAGATGTCCCATTCATCTCATGCCCTGATAAAAACTGATAAGGGGAGAGAATAGTTAAAAATTTTTCTAGGGTATCATAACTCTGGTAGGAAGTCATCTGTCTAGAAATCAAGAGAAAAAGAACGTGTGGCCTCCTGTTATAACAAGGGTTTCTAGATTTGTCCTGTGAAAGGTCGTTTAAGGACTTGGGGATCAACTTCCTCAATTATCACCAATTGCACTGTTGCTCCAAAAATCATTTAAAAGCTTACTGGACATATCTACATAATGGTGAAACTGTAATTTAGAGACTATCCCTGACTAATGTGCTGGTAGGCATTAAAATGAGTTCCCAAGGGAAGTGATTAAAATTTTTTTCTCTTCTGTTTTTTGAGAGAATTTCTAGATGTCCTGGGCCACAGTTAATTAAGATTTTTAGGGGGGACAGAAAGTTATACTGAAATCTTTAGAGCTCCCTTCCGCCGTTAAAATTATATATATATATATTTAAATTATACCTTAAGTTCTGGGGTACATGTGCAGAATGTGCAGGTTTGTTACATAGGTATACACGTGCCATGGTGGTTTGCGGCACCTGTCAACCCATCTACATTAGGTATTTCTCCTAATGCTCTCCCTCCCCTAGCCCCCCACCCCTGGACAGGCCCCATTGTGTGATGTTCCCCTCCCTGTGTCCATGTGTTTTCATTGTTCAACTCCCACTTCTAAGTGAGAACATGCGGTGTTTGGTTTTCTGTTCCTGTGTTAGTTTGCTGAGAATGATGGTTTCCAGGTTAAAATTATATATTTTTAAATAAATGAAAACTGTGTTTTTAAAAGAGGACTTTTGAGAAGTATATAGAAAAACCATTAATTTAGACTCTGTGAGATTAGGTTGCATGAAGAAGGTTTTCTGAATATTTGAAGAGTGGATAAATAAATGTCCCCCAAAGCAATAAAATCATAATCCTTTAAAATATAGGAAAAATAACTAATGGGAACTAGGCTTAATACTCGGGATGAAATAATCTGTACAACAAACTCCCATGACACATGTTTACCTATGTAACAAACCTGCACATGTACCCCTGAACTTAAAATAAAATTTAAAGTATAATAATAAAATAATATGGATTTTCTTT The following amino acid sequence<SEQ ID NO. 82> is the predicted amino acid sequence derived from theDNA sequence of SEQ ID NO. 81:MTGDFPSMPGHNTSRNSSCDPIVTPHLISLYFIVLIGGLVGVISILFLLVKMNTRSVTTMAVINLVVVHSVFLLTVPFRLTYLIKKTWMFGLPFCKFVSAMLHIHMYLTFLFYVVILVTRYLIFFKCKDKVEFYRKLHAVAASAGMWTLVIVIVVPLVVSRYGIHEEYNEEHCFKFHKELAYTYVKIINYMIVIFVIAVAVILLVFQVFIIMLMVQKLRHSLLSHQEFWAQLKNLFFIGVILVCFLPYQFFRIYYLNVVTHSNACNSKVAFYNEIFLSVTAISCYDLLLFVFGGSHWFKQKIIGLWNCVLCR The following DNA sequence nGPcR-40 <SEQ ID NO. 83>was identified in H. sapiens:GCAGGAGCACTGAAAATCAGGAACAATCCTGTATTTTTTGTGATAATCAACAAGGACAAAACTTCTCCATATGTAAATAACAGCGTTATGAGCAGCAATTCATCCCTGCTGGTGGCTGTGCAGCTGTGCTACGCGAACGTGAATGGGTCCTGTGTGAAAATCCCCTTCTCGCCGGGATCCCGGGTGATTCTGTACATAGTGTTTGGCTTTGGGGCTGTGCTGGCTGTGTTTGGAAACCTCCTGGTGATGATTTCAATCCTCCATTTCAAGCAGCTGCACTCTCCGACCAATTTTCTCGTTGCCTCTCTGGCCTGCGCTGATTTCTTGGTGGGTGTGACTGTGATGCCCTTCAGCATGGTCAGGACGGTGGAGAGCTGCTGGTATTTTGGGAGGAGTTTTTGTACTTTCCACACCTGCTGTGATGTGGCATTTTGTTACTCTTCTCTCTTTCACTTGTGCTTCATCTCCATCGACAGGTACATTGCGGTTACTGACCCCCTGGTCTATCCTACCAAGTTCACCGTATCTGTGTCAGGAATTTGCATCAGCGTGTCCTGGATCCTGCCCCTCATGTACAGCGGTGCTGTGTTCTACACAGGTGTCTATGACGATGGGCTGGAGGAATTATCTGATGCCCTAAACTGTATAGGAGGTTGTCAGACCGTTGTAAATCAAAACTGGGTGTTGACAGATTTTCTATCCTTCTTTATACCTACCTTTATTATGATAATTCTGTATGGTAACATATTTCTTGTGGCTAGACGACAGGCGAAAAAGATAGAAAATACTGGTAGCAAGACAGAATCATCCTCAGAGAGTTACAAAGCCAGAGTGGCCAGGAGAGAGAGAAAAGCAGCTAAAACCCTGGGGGTCACAGTGGTAGCATTTATGATTTCATGGTTACCATATAGCATTGATTCATTAATTGATGCCTTTATGGGCTTTATAACCCCTGCCTGTATTTATGAGATTTGCTGTTGGTGTGCTTATTATAACTCAGCCATGAATCCTTTGATTTATGCTTTATTTTACCCATGGTTTAGGAAAGCAATAAAAGTTATTGTAACTGGTCAGGTTTTAAAGAACAGTTCAGCAACCATGAATTTGTTTTCTGAACATATATAA The following amino acid sequence <SEQ IDNO. 84> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 83:MSSNSSLLVAVQLCYANVNGSCVKIPFSPGSRVILYIVFGFGAVLAVFGNLLVMISILHFKQLHSPTNFLVASLACADFLVGVTVMPFSMVRTVESCWYFGRSFCTFHTCCDVAFCYSSLFHLCFISIDRYIAVTDPLVYPTKFTVSVSGICISVSWILPLMYSGAVFYTGVYDDGLEELSDALNCIGGCQTVVNQNWVLTDFLSFFIPTFIMIILYGNIFLVARRQAKKIENTGSKTESSSESYKARVARRERKAAKTLGVTVVAFMISWLPYSIDSLIDAFMGFITPACIYEICCWCAYYNSAMNPLIYALFYPWFRKAIKVIVTGQVLKNSSATMNLFSEHI The following DNAsequence nGPCR-54 <SEQ ID NO. 85> was identified in H. sapiens:ACCATGAATGAGCCACTAGACTATTTAGCAAATGCTTCTGATTTCCCCGATTATGCAGCTGCTTTTGGAAATTGCACTGATGAAAACATCCCACTCAAGATGCACTACCTCCCTGTTATTTATGGCATTATCTTCCTCGTGGGATTTCCAGGCAATGCAGTAGTGATATCCACTTACATTTTCAAAATGAGACCTTGGAAGAGCAGCACCATCATTATGCTGAACCTGGCCTGCACAGATCTGCTGTATCTGACCAGCCTCCCCTTCCTGATTCACTACTATGCCAGTGGCGAAAACTGGATCTTTGGAGATTTCATGTGTAAGTTTATCCGCTTCAGCTTCCATTTCAACCTGTATAGCAGCATCCTCTTCCTCACCTGTTTCAGCATCTTCCGCTACTGTGTGATCATTCACCCAATGAGCTGCTTTTCCATTCACAAAACTCGATGTGCAGTTGTAGCCTGTGCTGTGGTGTGGATCATTTCACTGGTAGCTGTCATTCCGATGACCTTCTTGATCACATCAACCAACAGGACCAACAGATCAGCCTGTCTCGACCTCACCAGTTCGGATGAACTCAATACTATTAAGTGGTACAACCTGATTTTGACTGCAAGTACTTTCTGCCTCCCCTTGGTGATAGTGACACTTTGCTATACCACGATTATCCACACTTTGACCCATGGACTGCAAACTGACAGCTGCCTTAAGCAGAAAGCACGAAGGCTAACCATTCTGCTACTCCTTGCATTTTACGTATGTTTTTTACCCTTCCATATCTTGAGGGTCATTCAGGATCGAATCTCAGCCTGCTTTCAATCAGTTGTTCCATTGAGAATCAGATCCATGAAGCTTACATCGTTTCTAGACCATTATGCTGCTCTGAACACCTTTGGTAACCTGTTACTATATGTGGTGGTCAGCGACAACTTTCAGCAGGCTGTCTGCTCAACAGTGAGATGCAAAGTAAGCGGGAACCTTGAGCAAGCAAAGAAAATTAGTTACTCAAACAACCCTTGAThe following amino acid sequence <SEQ ID NO. 86> is the predicted aminoacid sequence derived from the DNA sequence of SEQ ID NO. 85:MNEPLDYLANASDFPDYAAAFGNCTDENIPLKMHYLPVIYGIIFLVGFPGNAVVISTYIFKMRPWKSSTIIMLNLACTDLLYLTSLPFLIHYYASGENWIFGDFMCKFIRFSFHFNLYSSILFLTCFSIFRYCVIIHPMSCFSIHKTRCAVVACAVVWIISLVAVIPMTFLITSTNRTNRSACLDLTSSDELNTIKWYNLILTASTFCLPLVIVTLCYTTIIHTLTHGLQTDSCLKQKARRLTILLLLAFYVCFLPFHILRVIQDRISACFQSVVPLRIRSMKLTSFLDHYAALNTFGNLLLYVVVSDNFQQAVCSTVRCKVSGNLEQAKKISYSN The following DNA sequencenGPCR-56 <SEQ ID NO. 87> was identified in H. sapiens:AAAAATTGCTGTACTGAACTATTGAATGGAACTTGGAAATAAAGTCCCTTCCAAAATAACTATTCTTCAACAGAGAGTAATAGGTAAATGTTTTAGAAGTGAGAGGACTCAAATTGCCAATGATTTACTCTTTTATTTTTCCTCCTAGGTTTCTGGGATAAGTATGTGCAAATAAAAAATAAACATGAGAAGGAACTGTAACCTGATTATGGATTTGGGAAAAAGATAAATCAACACACAAAGGGAAAAGTAAACTGATTGACAGCCCTCAGGAATGATGCCCTTTTGCCACAATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTGCTTCCCTGTACAGTTTAATGGTGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTATATCACACTTCAAACAACTTCATACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTGGGGTGTCTGGTCATGCCTTACAGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTAAAATTCACACAAGCACCGACATTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCGCTACTATGCTGTGTGTGATCCACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTCATTAGTTGGAGTGTCCCTGCTGTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAGAGATATATTACAAACATGTTCACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGACCTTTATGACTTCTTTTTATATACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAAGAACAGGCAAGATTAATTAGTGATGCCAATCAGAAGCTCCAAATTGGATTGGAAATGAAAAATGGAATTTCACAAAGCAAAGAAAGGAAAGCTGTGAAGACATTGGGGATTGTGATGGGAGTTTTCCTAATATGCTGGTGCCCTTTCTTTATCTGTACAGTCATGGACCCTTTTCTTCACTACATTATTCCACCTACTTTGAATGATGTA The following amino acidsequence <SEQ ID NO. 88> is the predicted amino acid sequence derivedfrom the DNA sequence of SEQ ID NO. 87:MMPFCHNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFLLGCLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNILVICVMIFISWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVYYRIYLIAKEQARLISDANQKLQIGLEMKNGISQSKERKAVKTLGIVMGVFLICWCPFFICTVMDPFLHYIIPPTLNDARGSRANSA The following DNA sequence nGPCR-56 <SEQ ID NO. 89> was identifiedin H. sapiens:GGAATGATGCCCTTTTGCCACAATATAATTAATATTTCCTGTGTGAAAAACAACTGGTCAAATGATGTCCGTGCTTCCCTGTACAGTTTAATGGTGCTCATAATTCTGACCACACTCGTTGGCAATCTGATAGTTATTGTTTCTATATCACACTTCAAACAACTTCATACCCCAACAAATTGGCTCATTCATTCCATGGCCACTGTGGACTTTCTTCTGGGGTGTCTGGTCATGCCTTACAGTATGGTGAGATCTGCTGAGCACTGTTGGTATTTTGGAGAAGTCTTCTGTAAAATTCACACAAGCACCGACATTATGCTGAGCTCAGCCTCCATTTTCCATTTGTCTTTCATCTCCATTGACCGCTACTATGCTGTGTGTGATCCACTGAGATATAAAGCCAAGATGAATATCTTGGTTATTTGTGTGATGATCTTCATTAGTTGGAGTGTCCCTGCTGTTTTTGCATTTGGAATGATCTTTCTGGAGCTAAACTTCAAAGGCGCTGAAGAGATATATTACAAACATGTTCACTGCAGAGGAGGTTGCTCTGTCTTCTTTAGCAAAATATCTGGGGTACTGACCTTTATGACTTCTTTTTATATACCTGGATCTATTATGTTATGTGTCTATTACAGAATATATCTTATCGCTAAAGAACAGGCAAGATTAATTAGTGATGCCAATCAGAAGCTCCAAATTGGATTGGAAATGAAAAATGGAATTTCACAAAGCAAAGAAAGGAAAGCTGTGAAGACATTGGGGATTGTGATGGGAGTTTTCCTAATATGCTGGTGCCCTTTCTTTATCTGTACAGTCATGGACCCTTTTCTTCACTACATTATTCCACCTACTTTGAATGATGTATTGATTTGGTTTGGCTACTTGAACTCTACATTTAATCCAATGGTTTATGCATTTTTCTATCCTTGGTTTAGAAAAGCACTGAAGATGATGCTGTTTGGTAAAATTTTCCAAAAAGATTCATCCAGGTGTAAATTATTTTTGGAATTGAGTTCATAG The following amino acid sequence <SEQ ID NO. 90> is the predictedamino acid sequence derived from the DNA sequence of SEQ ID NO. 89:MMPFCHNIINISCVKNNWSNDVRASLYSLMVLIILTTLVGNLIVIVSISHFKQLHTPTNWLIHSMATVDFLLGCLVMPYSMVRSAEHCWYFGEVFCKIHTSTDIMLSSASIFHLSFISIDRYYAVCDPLRYKAKMNILVICVMIFISWSVPAVFAFGMIFLELNFKGAEEIYYKHVHCRGGCSVFFSKISGVLTFMTSFYIPGSIMLCVYYRIYLIAKEQARLISDANQKLQIGLEMKNGISQSKERKAVKTLGIVMGVFLICWCPFFICTVMDPFLHYIIPPTLNDVLIWFGYLNSTFNPMVYAFFYPWFRKALKMMLFGKIFQKDSSRCKLFLELSS The following DNAsequence nGPCR-58 <SEQ ID NO. 91> was identified in H. sapiens:CTGTAAAGTAGATTGTATGAGGACTCCATGAGGTCATCCACTTCAAGTCCTTGGCATAGGATAATTACTCAAAAGGTGATGACAATGGCGCAGGGAGGGATGGTGACTTGCCTGGAGATGCACAGCACCGTCTCTCCCATACTCGGTCATTCACACCATCATTGATTCACCAGGCACCACTCCGTGTCCAGCAGGACTCTGGGGACCCCAAATGGACACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGATTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGCACCTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCATCCTACCCCAGGGGCCCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCGCCAGAGGCGGCCCCGGGCGCAGGCCCCACGTGAGGGTCCAGGAACACGCAGGCCCACCAGAGCAGTGAAAGAGCCCAGGGCAGACAGAGGAACCAGCCAGTCAGA The following amino acid seqence <SEQ ID NO. 92> isthe predicted amino acid sequence derived from the DNA sequence of SEQID NO. 91:LAWRCTAPSLPYSVIHTIIDSPGTTPCPAGLWGPQMDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFIMDVYSGYLLWEALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAASSVPSPCDEASPTPSSHPTPGALEDPATPPASEGESPSSTPPEAAPGAGPT Thefollowing DNA sequence nGPCR-58 <SEQ ID NO. 93> was identified in H.sapiens:ATGGACACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGACTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGCACCTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCATCCTACCCCAGGGGCCCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCGCCAGAGGCGGCCCCGGGCGCAGGCCCCACGTGA The following amino acid sequence <SEQID NO. 94> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 93:MDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAA The following DNAsequence nGPCR-3 <SEQ ID NO. 185> was identified in H. sapiens:AGGCTCGCGCCCGAAGCAGAGCCATGAGAACCCCAGGGTGCCTGGCGAGCCGCTAGCGCCATGGGCCCCGGCGAGGCGCTGCTGGCGGGTCTCCTGGTGATGGTACTGGCCGTGGCGCTGCTATCCAACGCACTGGTGCTGCTTTGTTGCGCCTACAGCGCTGAGCTCCGCACTCGAGCCTCAGGCGTCCTCCTGGTGAATCTGTCTCTGGGCCACCTGCTGCTGGCGGCGCTGGACATGCCCTTCACGCTGCTCGGTGTGATGCGCGGGCGGACACCGTCGGCGCCCGGCGCATGCCAAGTCATTGGCTTCCTGGACACCTTCCTGGCGTCCAACGCGGCGCTGAGCGTGGCGGCGCTGAGCGCAGACCAGTGGCTGGCAGTGGGCTTCCCACTGCGCTACGCCGGACGCCTGCGACCGCGCTATGCCGGCCTGCTGCTGGGCTGTGCCTGGGGACAGTCGCTGGCCTTCTCAGGCGCTGCACTTGGCTGCTCGTGGCTTGGCTACAGCAGCGCCTTCGCGTCCTGTTCGCTGCGCCTGCCGCCCGAGCCTGAGCGTCCGCGCTTCGCAGCCTTCACCGCCACGCTCCATGCCGTGGGCTTCGTGCTGCCGCTGGCGGTGCTCTGCCTCACCTCGCTCCAGGTGCACCGGGTGGCACGCAGACACTGCCAGCGCATGGACACCGTCACCATGAAGGCGCTCGCGCTGCTCGCCGACCTGCACCCCAGTGTGCGGCAGCGCTGCCTCATCCAGCAGAAGCGGCGCCGCCACCGCGCCACCAGGAAGATTGGCATTGCTATTGCGACCTTCCTCATCTGCTTTGCCCCGTATGTCATGACCAGGCTGGCGGAGCTCGTGCCCTTCGTCACCGTGAACGCCCAGTGGGGCATCCTCAGCAAGTGCCTGACCTACAGCAAGGCGGTGGCCGACCCGTTCACGTACTCTCTGCTCCGCCGGCCGTTCCGCCAAGTCCTGGCCGGCATGGTGCACCGGCTGCTGAAGAGAACCCCGCGCCCAGCATCCACCCATGACAGCTCTCTGGATGTGGCCGGCATGGTGCACCAGCTGCTGAAGAGAACCCCGCGCCCAGCGTCCACCCACAACGGCTCTGTGGACACAGAGAATGATTCCTGCCTGCAGCAGACACACTGAGGGCCTGGCAGGGCTCATCGCCCCCACCTTCTAAGA The following amino acid sequence <SEQ ID NO. 186>is the predicted amino acid sequence derived from the DNA sequence ofSEQ ID NO. 185: MGPGEALL AGLLVMVLAVALLSNALVLLCCA YSAELRTRASGVLLVNLSLGHLLLAALDMPFTLL GVMRGRTP SAPGAC QVIGFLDTFLASNAALSVAALSADQWLAVGFPLRYAGRLRPR YAGLLLGCAWGQSLAFSGAALGC SW LGYSSAFASCSLRLPPEPERPRFAAFTATLHAVGFVLPLAVLCLT SLQVHRVARRHCQRMDTVTMKALALLADLHPSVRQRCLIQQKRRRHRATR KIGIAIATFLICFAPYVMT RLAELVPFVTVNAQWGILSKCLTYSKAVADPF TYSLLRRPFRQVLAGMVHRLLKRTPRPASTHDSSLDVAGMVHQLLKRTPRPASTHNGSVDTENDSCLQQTH Thefollowing DNA sequence nGPCR-58 <SEQ ID NO. 93> was identified in H.sapiens:ATGGACACTACCATGGAAGCTGACCTGGGTGCCACTGGCCACAGGCCCCGCACAGAGCTTGATGATGAGGACTCCTACCCCCAAGGTGGCTGGGACACGGTCTTCCTGGTGGCCCTGCTGCTCCTTGGGCTGCCAGCCAATGGGTTGATGGCGTGGCTGGCCGGCTCCCAGGCCCGGCATGGAGCTGGCACGCGTCTGGCGCTGCTCCTGCTCAGCCTGGCCCTCTCTGACTTCTTGTTCCTGGCAGCAGCGGCCTTCCAGATCCTAGAGATCCGGCATGGGGGACACTGGCCGCTGGGGACAGCTGCCTGCCGCTTCTACTACTTCCTATGGGGCGTGTCCTACTCCTCCGGCCTCTTCCTGCTGGCCGCCCTCAGCCTCGACCGCTGCCTGCTGGCGCTGTGCCCACACTGGTACCCTGGGCACCGCCCAGTCCGCCTGCCCCTCTGGGTCTGCGCCGGTGTCTGGGTGCTGGCCACACTCTTCAGCGTGCCCTGGCTGGTCTTCCCCGAGGCTGCCGTCTGGTGGTACGACCTGGTCATCTGCCTGGACTTCTGGGACAGCGAGGAGCTGTCGCTGAGGATGCTGGAGGTCCTGGGGGGCTTCCTGCCTTTCCTCCTGCTGCTCGTCTGCCACGTGCTCACCCAGGCCACAGCCTGTCGCACCTGCCACCGCCAACAGCAGCCCGCAGCCTGCCGGGGCTTCGCCCGTGTGGCCAGGACCATTCTGTCAGCCTATGTGGTCCTGAGGCTGCCCTACCAGCTGGCCCAGCTGCTCTACCTGGCCTTCCTGTGGGACGTCTACTCTGGCTACCTGCTCTGGGAGGCCCTGGTCTACTCCGACTACCTGATCCTACTCAACAGCTGCCTCAGCCCCTTCCTCTGCCTCATGGCCAGTGCCGACCTCCGGACCCTGCTGCGCTCCGTGCTCTCGTCCTTCGCGGCAGCTCTCTGCGAGGAGCGGCCGGGCAGCTTCACGCCCACTGAGCCACAGACCCAGCTAGATTCTGAGGGTCCAACTCTGCCAGAGCCGATGGCAGAGGCCCAGTCACAGATGGATCCTGTGGCCCAGCCTCAGGTGAACCCCACACTCCAGCCACGATCGGATCCCACAGCTCAGCCACAGCTGAACCCTACGGCCCAGCCACAGTCGGATCCCACAGCCCAGCCACAGCTGAACCTCATGGCCCAGCCACAGTCAGACTCTGTGGCCCAGCCACAGGCAGACACTAACGTCCAGACCCCTGCACCTGCTGCCAGTTCTGTGCCCAGTCCCTGTGATGAAGCTTCCCCAACCCCATCCTCGCATCCTACCCCAGGGGCCCTTGAGGACCCAGCCACACCTCCTGCCTCTGAAGGAGAAAGCCCCAGCAGCACCCCGCCAGAGGCGGCCCCGGGCGCAGGCCCCACGTGA The following amino acid sequence <SEQID NO. 94> is the predicted amino acid sequence derived from the DNAsequence of SEQ ID NO. 93:MDTTMEADLGATGHRPRTELDDEDSYPQGGWDTVFLVALLLLGLPANGLMAWLAGSQARHGAGTRLALLLLSLALSDFLFLAAAAFQILEIRHGGHWPLGTAACRFYYFLWGVSYSSGLFLLAALSLDRCLLALCPHWYPGHRPVRLPLWVCAGVWVLATLFSVPWLVFPEAAVWWYDLVICLDFWDSEELSLRMLEVLGGFLPFLLLLVCHVLTQATACRTCHRQQQPAACRGFARVARTILSAYVVLRLPYQLAQLLYLAFLWDVYSGYLLWEALVYSDYLILLNSCLSPFLCLMASADLRTLLRSVLSSFAAALCEERPGSFTPTEPQTQLDSEGPTLPEPMAEAQSQMDPVAQPQVNPTLQPRSDPTAQPQLNPTAQPQSDPTAQPQLNLMAQPQSDSVAQPQADTNVQTPAPAA The following DNAsequence nGPCR-14 <SEQ ID NO. 191> was identified in H. sapiens:ACTAACTTTGGGAACTCGTATAGACCCAGCGTCGCTCCCCGCGCCGCCTCGCCTCCACTTTGGTTTCCCGCGTCCTGCCCGCCCTCTTCGGTGCCTCCTCTTCCTCCGGGACAAGGATGGAGGATCTCTTTAGCCCCTCAATTCTGCCGCCGGCGCCCAACATTTCCGTGCCCATCTTGCTGGGCTGGGGTCTCAACCTGACCTTGGGGCAAGGACCCCCTGCCTCTGGGCCGCCCAGCCCGCGTGCGGGGGCACGGCGCTGTCACAGCTGGCCTGGGAACTGCTGGGCGAGCCCCGCGCGGCCACGGGGGACCTGGCGTGCCGCTTCCTGCAGCTGCTGCAGGCATCCGGGCGGGGCGCCTCGGCCCACCTAGTGGTGCTCATCGCCCTCGAGCGCCGGCGCGCGGTGCGTCTTCCGCACGGCCGGCCGCTGCCCGCGCGTGCCCTCGCCGCCCTGGGCTGGCTGCTGGCACTGCTGCTGGCGCTGCCCCCGGCCTTCGTGGTGCGCGGGGACTCCCCCTCGCCGCTGCCGCCGCCGCCGCCGCCAACGTCCCTGCAGCCAGGCGCGCCCCCGGCCGCCCGCGCCTGGCCGGGGGAGCGTCGCTGCCACGGGATCTTCGCGCCCCTGCCGCGCTGGCACCTGCAGGTCTACGCGTTCTACGAGGCCGTCGCGGGCTTCGTCGCGCCTGTTACGGTCCTGGGCGTCGCTTGCGGCCACCTACTCTCCGTCTGGTGGCGGCACCGGCCGCAGGCCCCCGCGGCTGCAGCGCCCTGGTCGGCGAGCCCAGGTCGAGCCCCTGCGCCCAGCGCGCTGCCCCGCGCCAAGGTGCAGAGCCTGAAGATGAGCCTGCTGCTGGCGCTGCTGTTCGTGGGCTGCGAGCTGCCCTACTTTGCCGCCCGGCTGGCGGCCGCGTGGTCGTCCGGGCCCGCGGGAGACTGGGAGGGAGAGGGCCTGTCGGCGGCGCTGCGCGTGgTGGCGATGGCCAACAGCGCTCTCAATCCCTTCGTCTACCTCTTCTTCCAGGCGGGCGACTGCTGGCTCCGGCGACAGCTGCGGAAGCGGCTGGGCTCTCTGTGCTGCGCGCCGCAGGGAGGCGCGGAGGACGAGGAGGGGCCCCGGGGCCACCAGGCGCTCTACCGCCAACGCTGGCCCCACCCTCATTATCACCATGCTCGGCGGGAACCCGCTGGACGAGGGCGGCTTGCGCCCACCCCCTCCGCGCCCCAGACCCCTGCCTTGCTCCTGCGAAAGTGCCTTCTAGGTGCTTGGTGGTCAGAGACGGGTCATCTGTCGCTAAGGCGCAACCTCCAGGGAACTCGAGGCCTGCCAGGGTCTGTCCAGATCACAAGGGGCAGGAGAGTCTGTGAGAGAGTGACACTGAAGTTGTCCCCTTCCTCCACTCTCCTATTCCCTTCTCATGTTTACATTTCCCTATGCTCTTCCAGTTTCTCTTCTTCCCTACAGTTCCTCTCATATCTCCCCATTTGGAGACAGTGAGCCACTGGAAAGTTGTAAAAACAAAAACAGTTATTTTTGCAGTTTTCTTTCACGCATTTATAGTGCTCTGGATAATGCCATTTATTTTTGCTGATTACCCAACTTTCAGTATTTGCTGTGTTATCATCTGTATTTACTTATTTTGA The following amino acid sequence<SEQ ID NO. 192> is the predicted amino acid sequence derived from theDNA sequence of SEQ ID NO. 191:MEDLFSPSILPPAPNISVPILLGWGLNLTLGQGAPASGPPSRRVRLVFLGVILVVAVAGNTTVLCRLCGGGGPWAGPKRRKMDFLLVQLALADLYACGGTALSQLAWELLGEPRAATGDLACRFLQLLQASGRGASAHLVVLIALERRRAVRLPHGRPLPARALAALGWLLALLLALPPAFVVRGDSPSPLPPPPPPTSLQPGAPPAARAWPGERRCHGIFAPLPRWHLQVYAFYEAVAGFVAPVTVLGVACGHLLSVWWRHRPQAPAAAAPWSASPGRAPAPSALPRAKVQSLKMSLLLALLFVGCELPYFAARLAAAWSSGPAGDWEGEGLSAALRVVAMANSALNPFVYLFFQAGDCWLRRQLRKRLGSLCCAPQGGAEDEEGPRGHQALYRQRWPHPHYHHARREPAGRGRLAPTPSAPQTPALLLRKCLLGAWWSETGHLSLRRNLQGTRGLPGSVQITRGRRVCERVTLKLSPSSTLLFPSHVYISLCSSSFSSSLQFLSYLPIWRQ

Example 2 Cloning of nGPCR-x

To isolate a cDNA clone encoding full length nGPCR-x, a DNA fragmentcorresponding to a nucleotide sequence set forth in odd numberednucleotide sequences ranging from SEQ ID NO: 1-93, or a portion thereof,can be used as a probe for hybridization screening of a phage cDNAlibrary. The DNA fragment is amplified by the polymerase chain reaction(PCR) method. The PCR reaction mixture of 50 μl contains polymerasemixture (0.2 mM dNTPs, 1×PCR Buffer and 0.75 μl Expand High FidelityPolymerase (Roche Biochemicals)), 1 μg of plasmid, and 50 pmoles offorward primer and 50 pmoles of reverse primer. The primers arepreferably 10 to 25 nucleotides in length and are determined byprocedures well known to those skilled in the art. Amplification isperformed in an Applied Biosystems PE2400 thermocycler, using thefollowing program: 95° C. for 15 seconds, 52° C. for 30 seconds and 72°C. for 90 seconds; repeated for 25 cycles. The amplified product isseparated from the plasmid by agarose gel electrophoresis, and purifiedby Qiaquick™ gel extraction kit (Qiagen).

A lambda phage library containing cDNAs cloned into lambda ZAPIIphagovector is plated with E. coli XL-1 blue host, on 15 cm LB-agarplates at a density of 50,000 pfu per plate, and grown overnight at 37°C.; (plated as described by Sambrook et al., supra). Phage plaques aretransferred to nylon membranes (Amersham Hybond NJ), denatured for 2minutes in denaturation solution (0.5 M NaOH, 1.5 M NaCl), renatured for5 minutes in renaturation solution (1 M Tris pH 7.5, 1.5 M NaCl), andwashed briefly in 2×SSC (20×SSC: 3 M NaCl, 0.3 M Na-citrate). Filtermembranes are dried and incubated at 80° C. for 120 minutes tocross-link the phage DNA to the membranes.

The membranes are hybridized with a DNA probe prepared as describedabove. A DNA fragment (25 ng) is labeled with α-³²P-dCTP (NEN) usingRediprime™ random priming (Amersham Pharmacia Biotech), according tomanufacturers instructions. Labeled DNA is separated from unincorporatednucleotides by S200 spin columns (Amersham Pharmacia Biotech), denaturedat 95° C. for 5 minutes and kept on ice. The DNA-containing membranes(above) are pre-hybridized in 50 ml ExpressHyb™ (Clontech) solution at68° C. for 90 minutes. Subsequently, the labeled DNA probe is added tothe hybridization solution, and the probe is left to hybridize to themembranes at 68° C. for 70 minutes. The membranes are washed five timesin 2×SSC, 0.1% SDS at 42° C. for 5 minutes each, and finally washed 30minutes in 0.1×SSC, 0.2% SDS. Filters are exposed to Kodak XAR™ film(Eastman Kodak Company, Rochester, N.Y., USA) with an intensifyingscreen at −80° C. for 16 hours. One positive colony is isolated from theplates, and replated with about 1000 pfu on a 15 cm LB plate. Plating,plaque lift to filters and hybridization are performed as describedabove. About four positive phage plaques are isolated form thissecondary screening.

cDNA containing plasmids (pBluescript SK−) are rescued from the isolatedphages by in vivo excision by culturing XL-1 blue cells co-infected withthe isolated phages and with the Excision helper phage, as described bymanufacturer (Stratagene). XL-blue cells containing the plasmids areplated on LB plates and grown at 37° C. for 16 hours. Colonies (18) fromeach plate are replated on LB plates and grown. One colony from eachplate is stricken onto a nylon filter in an ordered array, and thefilter is placed on a LB plate to raise the colonies. The filter is thenhybridized with a labeled probe as described above. About three positivecolonies are selected and grown up in LB medium. Plasmid DNA is isolatedfrom the three clones by Qiagen Midi Kit™ (Qiagen) according to themanufacturer's instructions. The size of the insert is determined bydigesting the plasmid with the restriction enzymes NotI and SalI, whichestablishes an insert size. The sequence of the entire insert isdetermined by automated sequencing on both strands of the plasmids.

nGPCR-1: PCR and Subcloning

cDNAs were sequenced directly using an AB1377 fluorescence-basedsequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, FosterCity, Calif.) and the ABI PRISM Ready Dye-Deoxy Terminator kit with TaqFS polymerase. Each ABI cycle sequencing reaction contained about 0.5 μgof plasmid DNA. Cycle-sequencing was performed using an initialdenaturation at 98° C. for 1 min, followed by 50 cycles: 99° C. for 30sec, annealing at 50° C. for 30 sec, and extension at 60° C. for 4 min.Temperature cycles and times were controlled by a Perkin-Elmer 9600thermocycler. Extension products were purified using AGTC® gelfiltration block (Edge BiosSystems, Gaithersburg, Md.). Each reactionproduct was loaded by pipette onto the column, which was thencentrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at room temperature. Column-purifiedsamples were dried under vacuum for about 40 min and then dissolved in 5μl of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and1.6 mg/ml Blue Dextran). The samples were then heated to 90° C. forthree min and loaded into the gel sample wells for sequence analysis bythe ABI377 sequencer. Sequence analysis was performed by importingABI373A files into the Sequencher program (Gene Codes, Ann Arbor,Mich.).

The PCR reaction was performed in 50 μL samples containing 41.9 μL H₂O,5 μL 10× Buffer containing 15 mM MgCl₂ (Boehringer Mannheim Expand HighFidelity PCR System), 0.5 μL 10 mM dNTP mix, 1.5 μL human genomic DNA(Clontech #6550-1, 0.1 μg/μL), 0.3 μL primer VR1A (1 μg/μL), 0.3 μLprimer VR1B (1 μg/μL), and 0.5 μL High Fidelity Taq polymerase(Boehringer Mannheim, 3.5U/μl). The primer sequences for and,respectively were: 5′TCAAAGCTTATGGAATCATCTTTCTCATTTGGAGTGATCCTTGCTGTC,(VR1A) (SEQ ID NO: 95) corresponding to the 5′ end of the coding regionand containing a HindIII restriction site, and: 5′TTCACTCGAGTTAGCCATCAAACTCTGAGCTGGAGATAGTGACGATGTG (VR1B) (SEQ ID NO: 96)corresponding to the 3′ end of the coding region and containing an XhoIrestriction site (Genosys). The PCR reaction was carried out using aGeneAmp PCR9700 thermocycler (Perkin Elmer Applied Biosystems) andstarted with 1 cycle of 94° C. for 2 min followed by 5 cycles at 94° C.for 30 sec, 60° C. for 2 min, 72° C. for 1.5 min, followed by 20 cyclesat 94° C. for 30 sec, 60° C. for 30 sec, 72° C. for 1.5 min.

The PCR reaction was loaded onto a 0.75% agarose gel. The DNA band wasexcised from the gel and the DNA eluted from the agarose using aQIAquick gel extraction kit (Qiagen). The eluted DNA wasethanol-precipitated and resuspended in 4 μL H₂O for ligation. Theligation reaction consisted of 4 μL of fresh ethanol-precipitated PCRproduct and 1 μL of pCRII-TOPO vector (Invitrogen). The reaction wasgently mixed and allowed to incubate for 5 min. at room temperaturefollowed by the addition of 1 μL of 6×TOPO cloning stop solution andmixing for 10 sec. at room temperature. The sample was then placed onice and 2 μL was transformed in 50 μL of One Shot cells (Invitrogen) andplated onto ampicillin plates. Four white colonies were chosen and thepresence of an insert was verified by PCR in the following manner. Eachcolony was resuspended in 2 ml LB broth for 2 hrs. A 500 μL aliquot wasspun down in the microfuge, the supernatant discarded, and the pelletresuspended in 25 μL of H₂O. A 16 μL aliquot was removed and boiled for5 min and the sample was placed on ice. The sample was microfugedbriefly to pellet any bacterial debris and PCR was carried out with 15μL sample using primers VR1A and VR1B, described above.

Colonies from positive clones identified by PCR were used to inoculate a4 ml culture of LB medium containing 100 μg/ml ampicillin. Plasmid DNAwas purified using the Wizard Plus Minipreps DNA purification system(Promega). Since the primers used to amplify the fragment of nGPCR-1from genomic DNA were engineered to have HindIII and XhoI sites, thecDNA obtained from the minipreps was digested with these restrictionenzymes. One clone was verified by gel electrophoresis to give a DNAband of the correct size. cDNA from this clone was then sequenced,yielding the sequence of SEQ ID NO: 73.

nGPCR-3: PCR and Subcloning

First-strand cDNA synthesis was performed following the directions for3′-RACE ready cDNA from the SMART™ RACE cDNA Amplification Kit(Clontech). First 3 μl of H₂O, 1 μl human whole brain poly A⁺ RNA (1μg/μl) (Clontech, 6516-1) and 1 μl 3′-CDS primer were mixed together,incubated at 70° C. for 2 minutes, then placed on ice for 2 minutes.Added to the tube was 2 μl 5× First-Strand buffer, 1 μl 20 mM DTT, 1 μldNTP mix (10 mM) and 1 μl Superscript II RT (200 units/μl) (GIBCO/BRL).The tube was incubated at 42° C. for 1.5 hours then the reaction wasdiluted with 250 μl of Tricine-EDTA buffer.

PCR was performed in a 50 μl reaction using components that come withthe Advantage®-GC cDNA PCR Kit. The PCR reaction contained 22.4 μl H₂O,10 μl 5×GC cDNA PCR Reaction buffer, 10 μl 5M GC Melt, 1 μl 50×dNTP mix(10 mM each), 5 μl human brain cDNA, 0.3 μl of LW1649 (SEQ ID NO: 187)(1 μg/μl), 0.3 μl of LW1650 (SEQ ID NO: 188) (1 μg/μl), 1 μl 50×Advantage-GC cDNA polymerase mix. The PCR reaction was performed in aPerkin-Elmer 9600 GeneAmp PCR System starting with 1 cycle of 94° C. for2 min then 8 cycles at 94° C. for 15 sec, 72° C. for 2 min (decreasing1° C. with each cycle), 72° C. for 3 min, followed by 30 cycles of 94°C. for 15 sec, 68° C. for 3 min. The PCR reaction was loaded onto a 1.2%agarose gel. The DNA band was excised from the gel, placed in GenEluteAgarose spin column (Supelco) and spun for 10 min at maximum speed in amicrocentrifuge. The eluted DNA was EtOH precipitated and resuspended in4H₂O for ligation. The PCR primer sequence for LW1649 was:

GCATAAGCTTGCCATGGGCCCCGGCGAGG (SEQ ID NO: 187) and for LW1650 was:

GCATTCTAGACCTCAGTGTGTCTGCTGC (SEQ ID NO: 188). The underlined portion ofthe primers matches the 5′ and 3′ areas, respectively, of the codingregion.

The ligation reaction used solutions from the TOPO TA Cloning Kit(Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl Salt Solutionand 1 μl pCRII-TOPO vector that was incubated for 5 minutes at roomtemperature and then placed on ice. Two microliters of the ligationreaction was transformed in One-Shot TOP10 cells (Invitrogen), andplaced on ice for 30 minutes. The cells were heat-shocked for 30 secondsat 42° C., placed on ice for two minutes, 250 μl of SOC was added, thenincubated at 37° C. with shaking for one hour and then plated ontoampicillin plates. A single colony containing an insert was used toinoculate a 5 ml culture of LB medium. Plasmid DNA was purified using aConcert Rapid Plasmid Miniprep System (GibcoBRL) and then sequenced.

The DNA subcloned into pCRII-TOPO was sequenced using the ABI PRISM™ 310Genetic Analyzer (PE Applied Biosystems) which uses advanced capillaryelectrophoresis technology and the ABI PRISM™ BigDye™ Terminator CycleSequencing Ready Reaction Kit. Each cycle-sequencing reaction contained6 μl of H₂O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10sec, and 60° C. for 4 min. The product was purified using a Centriflex™gel filtration cartridge, dried under vacuum, then dissolved in 16 μl ofTemplate Suppression Reagent (PE Applied Biosystems). The samples wereheated at 95° C. for 5 min then placed in the 310 Genetic Analyzer,yielding the sequence of SEQ ID NO: 95.

nGPCR-9: PCR and Subcloning

The PCR reaction was performed in 50 μl containing 34.5 μl H₂O, 5 μlBuffer II (PE Applied Biosystems AmpliTaq Gold system), 6 μl 25 mMMgCl₂, 2 μl 10 mM dNTP mix, 1.5 μl human genomic DNA (Clontech #6550-1,0.1 μg/μl), 0.3 μl primer VR9A (1 μg/μl), 0.3 μl primer VR9B (1 μg/μl),and 0.4 μl AmpliTaq Gold™ DNA Polymerase. The primer sequences for VR9Aand VR9B were as follows:

VR9A 5′TTCAAAGCTTATGGAGTCGGGGCTGCTG 3′ (SEQ ID NO: 101), correspondingto the 5′ end of the coding region and containing a HindIII restrictionsite, and the reverse primer was:

VR9B 5′ TTCACTCGAGTCAGTCTGCAGCCGGTTCTG 3′, (SEQ ID NO: 102),corresponding to the 3′ end of the coding region and containing an XhoIrestriction site (Genosys). The PCR reaction was carried out using aGeneAmp PCR 9700 thermocycler (Perkin Elmer Applied Biosystems) andstarted with 1 cycle of 95° C. for 10 min, then 10 cycles at 95° C. for30 sec, 72° C. for 2 min decreasing 1° C. each cycle, 72° C. for 1 min.followed by 30 cycles at 95° C. for 30 sec, 60° C. for 30 sec, 72° C.for 1 min. The PCR reaction was loaded on a 0.75% gel. The DNA band wasexcised from the gel and the DNA was eluted from the agarose using aQIAquick gel extraction kit (Qiagen). The eluted DNA wasethanol-precipitated and resuspended in 4 μl H₂O for ligation. Theligation reaction consisted of 4 μl of fresh ethanol-precipitated PCRproduct and 1 μl of pCRII-TOPO vector (Invitrogen). The reaction wasgently mixed and allowed to incubate for 5 min at room temperaturefollowed by the addition of 1 μl of 6×TOPO cloning stop solution andmixing for 10 sec at room temperature. The sample was then placed on iceand 2 μl was transformed in 50 μl of One Shot cells (Invitrogen) andplated onto ampicillin plates. Five white colonies were chosen and wereused to inoculate a 4 ml culture of LB medium containing 100 μg/mlampicillin. Plasmid DNA was purified using the Wizard Plus Minipreps DNApurification system (Promega). Since the primers used to PCR SEQ-9 fromgenomic DNA were engineered to have HindIII and XhoI sites, the cDNAobtained from the minipreps was digested with these restriction enzymes.One clone was verified by gel electrophoresis to give a DNA band of thecorrect size. cDNA from this clone was then submitted for sequencing.One mutation was found (bp 621 T→G) and repaired as described as below.

The mutation in the identified clone was repaired using the QuikChangeSite-Directed Mutagenesis Kit (Stratagene). The PCR reaction contained39.3 μl H₂O, 5 μl 10× reaction buffer, 50 ng mini-prep cDNA, 1.25 μlprimer VR9E (100 ng/μl), 1.25 μl primer VR9F (100 ng/μl), 1 μl 20 mMdNTP mix, 1 μl Pfu DNA polymerase. The cycle conditions were 95° C. for30 sec, then 12 cycles at 95° C. for 30 sec, 55° C. for 1 min, 68° C.for 10 min. One μl of DpnI was added and the tube incubated at 37° C.for 1 hr. One μl of the DpnI-treated DNA was transformed into 50 μlEpicurian coli XL1-Blue supercompetent cells and the entire insert wasre-sequenced. The primer sequences used were:

VR9E: 5′ GCATCCTGGCCGCTATCTGTGCACTCTACG 3′ (SEQ ID NO: 103) and

VR9F: 5′ CGTAGAGTGCACAGATAGCGGCCAGGATGC 3′ (SEQ ID NO: 104)

where the base underlined was the base being corrected.

The clone described above was sequenced directly using an ABI377fluorescence-based sequencer (Perkin Elmer/Applied Biosystems Division,PE/ABD, Foster City, Calif.) and the ABI BigDye™ Terminator CycleSequencing Ready Reaction kit with Taq FS™ polymerase. Each ABI cyclesequencing reaction contained 0.5 μg of plasmid DNA. Cycle-sequencingwas performed using an initial denaturation at 98° C. for 1 min,followed by 50 cycles: 96° C. for 30 sec, annealing at 50° C. for 30sec, and extension at 60° C. for 4 min. Temperature cycles and timeswere controlled by a Perkin-Elmer 9600 thermocycler. Extension productswere purified using AGTC (R) gel filtration block (Edge BiosSystems,Gaithersburg, Md.). Each reaction product was loaded by pipette onto thecolumn, which was then centrifuged in a swinging bucket centrifuge(Sorvall model RT6000B tabletop centrifuge) at 1500×g for 4 min at roomtemperature. Column-purified samples were dried under vacuum for about40 min and then dissolved in 3 μl of a DNA loading solution (83%deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran). Thesamples were then heated to 90° C. for 3.5 min and loaded into the gelsample wells for sequence analysis by the ABI377 sequencer. Sequenceanalysis was performed by importing ABI377 files into the 310 GeneticAnalyzer, yielding the sequence of SEQ ID NO: 77.

nGPCR-11: PCR and Subcloning

PCR was performed in a 50 g reaction containing 32 μl H₂O, 5 A 10×TTbuffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-tricine pH8.4), 5 μl 15 mM MgSO₄, 2 μl 10 mM dNTP, 5 μl human genomic DNA (0.3μg/μL) (Clontech), 0.3 μl of LW1564 (1 μg/μl), 0.3 μl of LW1565 (1μg/μl), 0.4 μl High Fidelity Taq polymerase (Boehringer Mannheim). ThePCR reaction was performed in a GeneAmp 9600 PCR thermocycler (PEApplied Biosystems) starting with 1 cycle of 94° C. for 2 min followedby 17 cycles at 94° C. for 30 sec, 72° C. for 2 min decreasing 1° C.each cycle, 68° C. for 2 min, then 25 cycles of 94° C. for 30 sec, 55°C. for 30 sec, 68° C. for 2 min. The PCR reaction was loaded onto a 1.2%agarose gel. The DNA band was excised from the gel, placed in GenEluteAgarose spin column (Supelco) and spun for 10 min at maximum speed in amicrocentrifuge. The eluted DNA was EtOH precipitated and resuspended in4 μl H₂O for ligation. The forward PCR primer sequence was:

LW1564: GCATAAGCTTCCATGTACAACGGGTCGTGCTGC (SEQ ID NO: 107), and thereverse PCR primer was:

LW1565: GCATTCTAGATCAGTGCCACTCAACAATGTGGG (SEQ ID NO: 108).

The ligation reaction used solutions from the TOPO TA Cloning Kit(vitrogen) which consisted of 4 μl PCR product DNA and 1 μl pCkII-TOPOvector that was incubated for 5 minutes at room temperature. To theligation reaction one microliter of 6×TOPO Cloning Stop Solution wasadded then the reaction was placed on ice. Two microliters of theligation reaction was transformed in One Shot TOP10 cells (Invitrogen),and placed on ice for 30 minutes. The cells were heat-shocked for 30seconds at 42° C., placed on ice for two minutes, 250 Tl of SOC wasadded, then incubated at 37° C. with shaking for one hour and thenplated onto ampicillin plates. A single colony containing an insert wasused to inoculate a 5 ml culture of LB medium. Plasmid DNA was purifiedusing a Concert Rapid Plasmid Miniprep System (GibcoBRL) and thensequenced.

The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310Genetic Analyzer (PE Applied Biosystems) which uses advanced capillaryelectrophoresis technology and the ABI PRISM™ BigDye™ Terminator CycleSequencing Ready Reaction Kit. Each cycle-sequencing reaction contained6 μl of H₂O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1μg/μl), and 1 μl primer (25 ng/11) and was performed in a Perkin-Elmer9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10sec, and 60° C. for 4 min. The product was purified using a Centriflex™gel filtration cartridge, dried under vacuum, then dissolved in 16 μl ofTemplate Suppression Reagent (PE Applied Biosystems). The samples wereheated at 95° C. for 5 min then placed in the 310 Genetic Analyzer,yielding the sequence of SEQ ID NO: 79.

nGPCR-16: PCR and Subcloning

PCR was performed in a 50 μl reaction containing 32 μl H₂O, 5 μl 10×TTbuffer (140 mM Ammonium Sulfate, 0.1% gelatin, 0.6 M Tris-tricine pH8.4), 5 μl 15 mM MgSO₄, 2 μl 10 mM dNTP, 5 μl 2445704H1 DNA (0.17Tg/Tl), 0.3 μl of LW1587 (1 μg/μl), 0.3 μl of LW1588 (1 μg/μl), 0.4 μlHigh Fidelity Taq polymerase (Boehringer Mannheim). The PCR reaction wasperformed on a Robocycler thermocycler (Stratagene) starting with 1cycle of 94° C. for 2 min followed by 15 cycles of 94° C. for 30 sec,55° C. for 1.3 min, 68° C. for 2 min. The PCR reaction was loaded onto a1.2% agarose gel. The DNA band was excised from the gel, placed inGenElute Agarose spin column (Supelco) and spun for 10 min at maximumspeed in a microcentrifuge. The eluted DNA was EtOH precipitated andresuspended in 12 μl H₂O for ligation. The PCR primer sequence for theforward primer was:

LW1587: GATCAAGCTTATGACAGGTGACTTCCCAAGTATGC (SEQ ID NO: 111), and thesequence for the reverse primer was:

LW1588: GATCCTCGAGGCTAACGGCACAAAACACAATTCC (SEQ ID NO: 112).

The ligation reaction used solutions from the TOPO TA Cloning Kit(Invitrogen) which consisted of 4 μl PCR product DNA and 1 μl pCRII-TOPOvector that was incubated for 5 minutes at room temperature. To theligation reaction one microliter of 6×TOPO Cloning Stop Solution wasadded then the reaction was placed on ice. Two microliters of theligation reaction was transformed in One-Shot TOP10 cells (Invitrogen),and placed on ice for 30 minutes. The cells were heat-shocked for 30seconds at 42° C., placed on ice for two minutes, 250 μl of SOC wasadded, then incubated at 37° C. with shaking for one hour and thenplated onto ampicillin plates. A single colony containing an insert wasused to inoculate a 5 ml culture of LB medium. Plasmid DNA was purifiedusing a Concert Rapid Plasmid Miniprep System (GibcoBRL) and thensequenced.

The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310Genetic Analyzer (PE Applied Biosystems) which uses advanced capillaryelectrophoresis technology and the ABI PRISM™ BigDye™ Terminator CycleSequencing Ready Reaction Kit. Each cycle-sequencing reaction contained6 μl of H₂O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10sec, and 60° C. for 4 min. The product was purified using a Centriflex™gel filtration cartridge, dried under vacuum, then dissolved in 16 μl ofTemplate Suppression Reagent (PE Applied Biosystems). The samples wereheated at 95° C. for 5 min then placed in the 310 Genetic Analyzer,yielding the sequence of SEQ ID NO: 81.

nGPCR-40: PCR and Subcloning

PCR was performed in a 50 μl reaction containing utilizing Herculase DNAPolymerase blend (Stratagene), using the buffer recommendations providedby the manufacturer, 200 ng each of primers PSK 18 and 19 (SEQ ID NOS:115 and 116), 150 ng of human genomic DNA (Clontech), and 2% DMSO. ThePCR reaction was performed on a Robocycler thermocycler (Stratagene)starting with 1 cycle of 94° C. for 2 min followed by 35 cycles of 94°C. for 30 sec, 65° C. for. 30 sec, 72° C. for 2 min. The PCR reactionwas purified using the QiaQuick PCR Purification Kit (Qiagen), and theneluted in TE. The PCR primer sequences were:

PSK 18 GATC GAATTCGCAGGAGCAATG AAAATCAGGAAC (SEQ ID NO: 115), and:

PSK19: GATCGAATTCTTATATATGTTCAGAAAACAAATTCATGG (SEQ ID NO: 116)). Theunderlined portion of the primer matches the 5′ and 3′ areas,respectively, of a portion of the 5′ untranslated region and codingregion. Initiation and termination codons are shown above in bold.

The PCR product was ligated into the pCR-BluntII-TOPO vector(Invitrogen) using the Zero Blunt Topo PCR TA cloning kit as follow: 3μl PCR product DNA, 1 μl pCRII-TOPO vector, and 1 μl TOPOII saltsolution (1.2M NaCl, 0.06M MgCl₂). The mixture was incubated for 5minutes at room temperature. To the ligation reaction one microliter of6×TOPO Cloning Stop Solution was added, and then the reaction was placedon ice. Two microliters of the ligation reaction was transformed inOne-Shot TOP10 cells (Invitrogen), and placed on ice for 30 minutes. Thecells were heat-shocked for 30 seconds at 42° C., placed on ice for twominutes, 250 μl of SOC was added, then incubated at 37° C. with shakingfor one hour and then plated onto ampicillin plates supplemented withXgal and IPTG. Single colonies were screened by PCR for the presence ofthe insert, and a plasmid DNA from colony 58 was purified using a QiagenEndo-Free plasmid purification kit.

nGPCR-40 was sequenced directly using an ABI377 fluorescence-basedsequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, FosterCity, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing ReadyReaction kit with Taq FS™ polymerase. Each ABI cycle sequencing reactioncontained about 0.5 μg of plasmid DNA. Cycle-sequencing was performedusing an initial denaturation at 98° C. for 1 min, followed by 50cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extensionat 60° C. for 4 min. Temperature cycles and times were controlled by aPerkin-Elmer 9600 thermocycler. Extension products were purified usingAGTC® gel filtration block (Edge BiosSystems, Gaithersburg, Md.). Eachreaction product was loaded by pipette onto the column, which was thencentrifuged in a swinging bucket centrifuge (Sorvall model RT6000Btabletop centrifuge) at 1500×g for 4 min at room temperature.Column-purified samples were dried under vacuum for about 40 min andthen dissolved in 3 μl of DNA loading solution (83% deionized formamide,8.3 mM EDTA, and 1.6 mg/ml Blue Dextran). The samples were then heatedto 90° C. for 3.5 min and loaded into the gel sample wells for sequenceanalysis by the ABI377 sequencer. Sequence analysis was performed byimporting ABI377 files into the Sequencher program (Gene Codes, AimArbor, M), which yielded a sequence identical to SEQ ID NO:83 with theexception that the nucleotide at position 10 was identified as an “A”which incorrectly indicated the presence of an initiation codon at thatposition. Subsequent analysis of genomic DNA samples indicated that thisposition was incorrectly assigned and that the correct nucleotide atthat position was a “C”. The sequence reported at SEQ ID NO. 83correctly identifies the nucleotide at position 10 and indicates thatthe first initiation codon occurs at position 88-90.

nGPCR-54: PCR and Subcloning

Two microliters of a human genomic library (˜10⁸ PFU/ml) (Clontech) wasadded to 6 ml of an overnight culture of K802 cells (Clontech), thendistributed as 250 μl aliquots into each of 24 tubes. The tubes wereincubated at 37° C. for 15 min. Seven milliliters of 0.8% agarose wasadded to each tube, mixed, then poured onto LB agar+10 mM MgSO₄ platesand incubated overnight at 37° C. To each plate 5 ml of SM (0.1M NaCl,8.1 mM MgSO₄-7H₂O, 50 mM Tris-Cl (pH 7.5), 0.0001% gelatin) phage bufferwas added and the top agarose was removed with a microscope slide andplaced in a 50 ml centrifuge tube. A drop of chloroform was added andthe tube was place in a 37° C. shaker for 15 min, then centrifuged for20 min at 4000 RPM (Sorvall RT6000 table top centrifuge) and thesupernatant stored at 4° C. as a stock solution.

Two μl of phage from each tube was heated to 99° C. for 4 min thencooled to 10° C. Added to the phage was a PCR mix containing 8.8 μl H₂O,4 μl 5× Rapid-load Buffer (Origene), 2 μl 10×PCR buffer II(Perkin-Elmer), 2 μl 25 mM MgCl₂, 0.8 μl 10 mM dNTP, 0.12 μl LW1634 (1μg/μl) (SEQ ID NO: 119), 0.12 μl LW1635 (1 μg/μl) (SEQ ID NO: 120), 0.2μl AmpliTaq Gold polymerase (Perlin Elmer). The PCR reaction involved 1cycle at 95° C. for 10 min followed by 35 cycles at 95° C. for 45 sec,53.5° C. for 2 min, 72° C. for 45 sec. The reaction was loaded onto a 2%agarose gel. From the tube that gave a PCR product of the correct size,10 μl was used to set up five 1:10 dilutions that were plated onto LBagar+10 mM MgSO₄ plates and incubated overnight. A BA85 nitrocellulosefilter (Schleicher & Schuell) was placed on top of each plate for 1hour. The filter was removed, placed phage side up in a petri dish, andcovered with 4 ml of SM for 15 min to elute the phage. One milliliter ofSM was removed from each plate and used to set up a PCR reaction asabove. The plate of the lowest dilution to give a PCR product wassubdivided, filter-lifted and the PCR reaction was repeated. The seriesof dilutions and subdividing of the plate was continued until a singleplaque was isolated that gave a positive PCR band. Once a single plaquewas isolated, 10 μl phage supernatant was added to 100 μl SM and 200 μlof K802 cells per plate with a total of 8 plates set up. The plates wereincubated overnight at 37° C. The top agarose was removed by adding 8 mlof SM then scrapping off the agarose with a microscope slide andcollected in a centrifuge tube. To the tube, 3 drops of chloroform wasadded, vortexed, incubated at 37° C. for 15 min then centrifuged for 20min at 4000 RPM (Sorvall RT6000 table top centrifuge) to recover thephage, which was used to isolate genomic phage DNA using the QiagenLambda Midi Kit. The sequence for primer LW1634 was:

CTGAAAGTTGTCGCTGACC (SEQ ID NO: 119), and for primer LW1635 was:

CGATTATCCACACTTTGACCC (SEQ ID NO: 120).

The PCR reaction for the coding region was performed in a 50 μl reactioncontaining 33 μl H₂O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1%gelatin, 0.6 M Tris tricine pH 8.4), 5 μl 15 mM MgSO₄, 2 μl 10 mM DNTP,4 μl genomic phage DNA (0.25 μg/μl), 0.3 μl LW1698 (1 μg/μl) (SEQ ID NO:121), 0.3 μl LW1699 (1 μg/μl) (SEQ ID NO: 122), 0.4 μl High Fidelity Taqpolymerase (Boehringer Mannheim). The PCR reaction was started with 1cycle of 94° C. for 2 min followed by 30 cycles at 94° C. for 30 sec,55° C. for 30 sec., 68° C. for 2 min. The PCR reaction was loaded onto a2% agarose gel. The DNA band was excised from the gel, placed inGenElute Agarose spin column (Supelco) and spun for 10 min at maximumspeed. The eluted DNA was EtOH precipitated and resuspended in 8 μl H₂O.The PCR primer sequence for primer LW1698 was:

GCATACCATGAATGAGCCACTAGAC (SEQ ID NO: 121), and for primer LW1699 was:

GCATCTCGAGTCAAGGGTTGTTTGAGTAAC (SEQ ID NO: 122). The underlined portionof the primer matches the 5′ and 3′ areas, respectively, of the codingregion of nGPCR-54.

The ligation reaction used solutions from the TOPO TA Cloning Kit(Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl of saltsolution and 1 μl pCRII-TOPO vector that was incubated for 5 minutes atroom temperature then the reaction was placed on ice. Two microliters ofthe ligation reaction was transformed in One-Shot TOP10 cells(Invitrogen), and placed on ice for 30 minutes. The cells wereheat-shocked for 30 seconds at 42° C., placed on ice for two minutes,250 μl of SOC was added, then incubated at 37° C. with shaking for onehour and then plated onto ampicillin plates. A single colony containingan insert was used to inoculate a 5 ml culture of LB medium. Plasmid DNAwas purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL)and then sequenced.

nGPCR-54 genomic phage DNA was sequenced using the ABI PRISM™ 310Genetic Analyzer (PE Applied Biosystems) which uses advanced capillaryelectrophoresis technology and the ABI PRISM™ BigDye™ Terminator CycleSequencing Ready Reaction Kit. The cycle-sequencing reaction contained14 μl of H₂O, 16 μl of BigDye Terminator mix, 7 μl genomic phage DNA(0.1 μg/μl), and 3 μl primer (25 ng/μl). The reaction was performed in aPerkin-Elmer 9600 thermocycler at 95° C. for 5 min, followed by 99cycles of 95° C. for 30 sec, 55° C. for 20 sec, and 60° C. for 4 min.The product was purified using a Centriflex™ gel filtration cartridges,dried under vacuum, then dissolved in 16 μl of Template SuppressionReagent. The samples were heated at 95° C. for 5 min then placed in the310 Genetic Analyzer.

The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310Genetic Analyzer (PE Applied Biosystems) which uses advanced capillaryelectrophoresis technology and the ABI PRISM™ BigDye™ Terminator CycleSequencing Ready Reaction Kit. Each cycle-sequencing reaction contained6 μl of H₂O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10sec, and 60° C. for 4 min. The product was purified using a Centriflex™gel filtration cartridge, dried under vacuum, then dissolved in 16 μl ofTemplate Suppression Reagent (PE Applied Biosystems). The samples wereheated at 95° C. for 5 min then placed in the 310 Genetic Analyzer,yielding the sequence of SEQ ID NO: 85.

nGPCR-56: PCR and Subcloning

The PCR reaction for the coding region of nGPCR-56 used components thatcome with PLATINUM® Pfx DNA Polymerase (GibcoBRL) containing 35.5 μlH₂O, 5 μl 10×Pfx Amplification buffer, 1.5 μl 50 mM MgSO₄, 2 μl 10 mM&M, 5 μl human genomic DNA (0.3 μg/μl) (Clontech), 0.3 μl of LW1603 (1μg/μl) (SEQ ID NO: 152), 0.3 μl of LW1604 (1 μg/μl) (SEQ ID NO: 153),0.4 μl PLATINUM® Pfa DNA Polymerase (2.5 U/Tl). The PCR reaction wasperformed in a Robocycler Gradient 96 (Stratagene) starting with 1 cycleof 94° C. for 5 min followed by 30 cycles at 94° C. for 40 sec, 55° C.for 2 min, 68° C. for 3 min. Following the final cycle, 0.5 μl ofAmpliTaq DNA Polymerase (5 U/μl) was added and the tube was incubated at72° C. for 5 min. The sequence of LW1603 is:

GATCAAGCTTGGAATGATGCCCTITTGCCAC (SEQ ID NO: 152), and for LW1604 is:

GATCCTCGAGCATCATTCAAAGTAGGTGG. (SEQ ID NO: 153). The underlined portionof the primer matches the 5′ and 3′ areas, respectively, of a portion ofthe coding region of nGPCR-56.

The PCR reaction for the coding region was performed in a 50 μl reactioncontaining 32 μl H₂O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1%gelatin, 0.6 M Tris tricine pH 8.4), 5 μl 15 mM MgSO₄, 2 μl 10 mM DNTP,5 μl human genomic DNA (0.3 μg/μl) (Clontech), 0.3 μl LW1603 (1 μg/μl)(SEQ ID NO: 152), 0.3 μl LW1696 (1 μg/μl) (SEQ ID NO: 154), 0.4 μl HighFidelity Taq polymerase (Boehringer Mannheim). The PCR reaction wasstarted with 1 cycle of 94° C. for 2 min followed by 25 cycles at 94° C.for 40 sec, 55° C. for 60 sec., 68° C. for 2 min. The PCR reaction wasloaded onto a 2% agarose gel. The DNA band was excised from the gel,placed in GenElute Agarose spin column (Supelco) and spun for 10 min atmaximum speed. The eluted DNA was EtOH precipitated and resuspended in12 μl H₂O for ligation. The PCR primer sequence for LW1603 is:

GATCAAGCTTGGAATGATGCCCTTTTGCCAC (SEQ ID NO: 152), and LW1696:

GATCCTCGAGCTATGAACTCAATTCCAAAAATAATTTACACC (SEQ ID NO: 154). Theunderlined portion of the primer matches the 5′ and 3′ areas,respectively, of a portion of the coding region.

The ligation reaction used solutions from the TOPO TA Cloning Kit(Invitrogen) which consisted of 4 μl PCR product DNA, 1 μl of saltsolution and 1 μl pCRH-TOPO vector that was incubated for 5 minutes atroom temperature then the reaction was placed on ice. Two microliters ofthe ligation reaction was transformed in One-Shot TOP10 cells(Invitrogen), and placed on ice for 30 minutes. The cells wereheat-shocked for 30 seconds at 42° C., placed on ice for two minutes,250 μl of SOC was added, then incubated at 37° C. with shaking for onehour and then plated onto ampicillin plates. A single colony containingan insert was used to inoculate a 5 ml culture of LB medium. Plasmid DNAwas purified using a Concert Rapid Plasmid Miniprep System (GibcoBRL)and then sequenced.

The mutation in nGPCR-56 was repaired using the QuikChange Site-DirectedMutagenesis Kit (Stratagene). The PCR reaction contained 40 μl H₂O, 5 μl10× Reaction buffer, 1 μl mini-prep DNA, 1 μl LW1700 (125 ng/μl) (SEQ IDNO: 155), 1 μl LW1701 (125 ng/μl) (SEQ ID NO: 156), 1 μl 10 mM dNTP, 1μl Pfu DNA polymerase. The cycle conditions were 95° C. for 30 sec then14 cycles at 95° C. for 30 sec, 55° C. for 1 min, 68° C. for 12 min. Thetube was placed on ice for 2 min, then 1 μl of DpnI was added and thetube incubated at 37° C. for one hour. One microliter of theDpnI-treated DNA was transformed into Epicurian coli XL1-Bluesupercompetent cells and the entire insert was re sequenced. The primersequences are:

-   GCTACTTGAACTCTACATTTAATCCAATGGTTTATGCATITTTCTATCC (LW1700) (SEQ ID    NO: 155), and:-   GGATAGAAAAATGCATAAACCATTGGATTAAATGTAGAGTTCAAGTAGC (LW1701) (SEQ ID    NO: 156).

The DNA subcloned into pCRII was sequenced using the ABI PRISM™ 310Genetic Analyzer (PE Applied Biosystems) which uses advanced capillaryelectrophoresis technology and the ABI PRISM™ BigDye™ Terminator CycleSequencing Ready Reaction Kit. Each cycle-sequencing reaction contained6 μl of H₂O, 8 μl of BigDye Terminator mix, 5 μl mini-prep DNA (0.1μg/μl), and 1 μl primer (25 ng/μl) and was performed in a Perkin-Elmer9600 thermocycler with 25 cycles of 96° C. for 10 sec, 50° C. for 10sec, and 60° C. for 4 min. The product was purified using a Centriflex™gel filtration cartridge, dried under vacuum, then dissolved in 16 μl ofTemplate Suppression Reagent (PE Applied Biosystems). The samples wereheated at 95° C. for 5 min then placed in the 310 Genetic Analyzer,yielding the sequence of SEQ ID NO: 89.

nGPCR-58: PCR and Subcloning

Isolation of a clone for nGPCR-58 from genomic DNA was performed by PCRin a 50 μl reaction containing Herculase DNA Polymerse blend(Stratagene), with buffer recommendations as supplied by themanufacturer, 200 ng each primers PSK14 (SEQ ID NO: 157) and PSK15 (SEQID NO: 158), 150 ng of human genomic DNA (Clontech) and 6% DMSO. The PCRreaction was performed on a Robocycler thermocycler (Stratagene)starting with 1 cycle of 94° C. for 2 min followed by 35 cycles of 94°C. for 30 sec, 65° C. for 30 sec, 72° C. for 2 min. The PCR reaction waspurified by the QiaQuick PCR Purification Kit (Qiagen) and eluted in Th.The PCR primer sequences were:

PSK14: 5′GATCGAATTCATGGACACTACCATGGAAGCTGACC (SEQ ID NO: 157), and:

PSK15: 5′GATCCTCGAGTCACGTGGGGCCTGCGCCCGG (SEQ ID NO: 158).

The underlined portion of the primers match the 5′ and 3′ areas,respectively, of a portion of the 5′ untranslated region and codingregion. Translation initiation and termination codons are shown above inbold.

The blunt ended PCR product was prepared for cloning by the addition ofa single base “A” residue by AmpliTaq Gold (Perkin Elmer) in a reactionwith 1×PCR Buffer II, 1 mM MgCl₂, 200 uM each dATP, dGTP, dCTP, anddTTP. The reaction was incubated at 94° C. for 10 minutes followed by72° C. for 10 minutes. The products were cloned into the pCRII-TOPOvector (Invitrogen) using the TOPO TA cloning kit as follows: 3 μl PCRproduct DNA, 1 μl pCRII-TOPO vector, and 1 μl TOPOII salt solution (1.2MNaCl, 0.06M MgCl₂) was incubated for 5 minutes at room temperature. Tothe ligation reaction one microliter of 6×TOPO Cloning Stop Solution wasadded then the reaction was placed on ice. Two microliters of theligation reaction was transformed in One-Shot TOP10 cells (Invitrogen),and placed on ice for 30 minutes. The cells were heat-shocked for 30seconds at 42° C., placed on ice for two minutes, 250 μl of SOC wasadded, then incubated at 37° C. with shaking for one hour and thenplated onto ampicillin plates supplemented with X-gal and IPTG. Singlecolonies were screened by PCR for the presence of the insert, and aplasmid DNA from colony 58-6 was purified using a Qiagen Endo-Freeplasmid purification kit and deposited as nGPCR-58.

nGPCR-58 was sequenced directly using an ABI377 fluorescence-basedsequencer (Perkin Elmer/Applied Biosystems Division, PE/ABD, FosterCity, Calif.) and the ABI BigDye™ Terminator Cycle Sequencing ReadyReaction kit with Taq FS™ polymerase. Each ABI cycle sequencing reactioncontained about 0.5 μg of plasmid DNA. Cycle-sequencing was performedusing an initial denaturation at 98° C. for 1 min, followed by 50cycles: 96° C. for 30 sec, annealing at 50° C. for 30 sec, and extensionat 60° C. for 4 min. Temperature cycles and times were controlled by aPerkin-Elmer 9600 thermocycler. Extension products were purified usingAGTC (R) gel filtration block (Edge RiosSystems, Gaithersburg, Md.).Each reaction product was loaded by pipette onto the column, which wasthen centrifuged in a swinging bucket centrifuge (Sorvall model RT6000Btabletop centrifuge) at 1500×g for 4 min at room temperature.Column-purified samples were dried under vacuum for about 40 min andthen dissolved in 3 μl of a DNA loading solution (83% deionizedformamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran). The samples werethen heated to 90° C. for 3.5 min and loaded into the gel sample wellsfor sequence analysis by the ABI377 sequencer. Sequence analysis wasperformed by importing ABI377 files into the Sequencer program (GeneCodes, Ann Arbor, Mich.), yielding the sequence of SEQ ID NO: 93.

Example 3 Hybridization Analysis to Demonstrate nGPCR-X Expression inBrain

The expression of nGPCR-x in mammals, such as the rat, may beinvestigated by in situ hybridization histochemistry. To investigateexpression in the brain, for example, coronal and sagittal rat braincryosections (20 pun thick) are prepared using a Reichert-Jung cryostat.Individual sections are thaw-mounted onto silanized, nuclease-freeslides (CEL Associates, Inc., Houston, Tex.), and stored at −80° C.Sections are processed starting with post-fixation in cold 4%paraformaldehyde, rinsed in cold phosphate-buffered saline (PBS),acetylated using acetic anhydride in triethanolamine buffer, anddehydrated through a series of alcohol washes in 70%, 95%, and 100%alcohol at room temperature. Subsequently, sections are delipidated inchloroform, followed by rehydration through successive exposure to 100%and 95% alcohol at room temperature. Microscope slides containingprocessed cryosections are allowed to air dry prior to hybridization.Other tissues may be assayed in a similar fashion.

A nGPCR-x-specific probe is generated using PCR. Following PCRamplification, the fragment is digested with restriction enzymes andcloned into pBluescript II cleaved with the same enzymes. For productionof a probe specific for the sense strand of nGPCR-x, the nGPCR-x clonein pBluescript II is linearized with a suitable restriction enzyme,which provides a substrate for labeled run-off transcripts (i.e., cRNAriboprobes) using the vector-borne T7 promoter and commerciallyavailable T7 RNA polymerase. A probe specific for the antisense strandof nGPCR-x is also readily prepared using the nGPCR-x clone inpBluescript II by cleaving the recombinant plasmid with a suitablerestriction enzyme to generate a linearized substrate for the productionof labeled run-off cRNA transcripts using the T3 promoter and cognatepolymerase. The riboprobes are labeled with [³⁵S]-UTP to yield aspecific activity of about 0.40×10⁶ cpm/pmol for antisense riboprobesand about 0.65×10⁶ cpm/pmol for sense-strand riboprobes. Each riboprobeis subsequently denatured and added (2 pmol/ml) to hybridization bufferwhich contained 50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH8.0), 1 mM EDTA, 1× Denhardt's Solution, and 10 mM dithiothreitol.Microscope slides containing sequential brain cryosections areindependently exposed to 45 μl of hybridization solution per slide andsilanized cover slips are placed over the sections being exposed tohybridization solution. Sections are incubated overnight (15-18 hours)at 52° C. to allow hybridization to occur. Equivalent series ofcryosections are exposed to sense or antisense nGPCR-40-specific cRNAriboprobes.

Following the hybridization period, coverslips are washed off the slidesin 1×SSC, followed by RNase A treatment involving the exposure of slidesto 20 μg/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5M EDTA, and 0.5 M NaCl for 45 minutes at 37° C. The cryosections arethen subjected to three high-stringency washes in 0.1×SSC at 52° C. for20 minutes each. Following the series of washes, cryosections aredehydrated by consecutive exposure to 70%, 95%, and 100% ammoniumacetate in alcohol, followed by air drying and exposure to Kodak BioMax™MR-1 film. After 13 days of exposure, the film is developed. Based onthese results, slides containing tissue that hybridized, as shown byfilm autoradiograms, are coated with Kodak NTB-2 nuclear track emulsionand the slides are stored in the dark for 32 days. The slides are thendeveloped and counterstained with hematoxylin. Emulsion-coated sectionsare analyzed microscopically to determine the specificity of labeling.The signal is determined to be specific if autoradiographic grains(generated by antisense probe hybridization) are clearly associated withcresyl violate-stained cell bodies. Autoradiographic grains foundbetween cell bodies indicates non-specific binding of the probe.

Expression of nGPCR-x in the brain provides an indication thatmodulators of nGPCR-x activity have utility for treating neurologicaldisorders, including but not limited to, schizophrenia, affectivedisorders, ADHD/ADD (i.e., Attention Deficit-HyperactivityDisorder/Attention Deficit Disorder), and neural disorders such asAlzheimer's disease, Parkinson's disease, migraine, and senile dementia.Some other diseases for which modulators of nGPCR-x may have utilityinclude depression, anxiety, bipolar disease, epilepsy, neuritis,neurasthenia, neuropathy, neuroses, and the like. Use of nGPCR-xmodulators, including nGPCR-x ligands and anti-nGPCR-x antibodies, totreat individuals having such disease states is intended as an aspect ofthe invention.

Example 4 Tissue Expression Profiling

Tissue specific expression of the cDNAs encoding nGPCR-1, nGPCR-3,nGPCR-9, nGPCR-11, nGPCR-16, nGPCR-40, nGPCR-54, nGPCR-56, and nGPCR-58was detected using a PCR-based system. Tissue specific expression ofcDNAs encoding nGPCR-x may be accomplished using similar methods.

Primers were synthesized by Genosys Corp., The Woodlands, Tex. PCRreactions were assembled using the components of the Expand Hi-Fi PCRSystem™ (Roche Molecular Biochemicals, Indianapolis, Ind.).

nGPCR-1

The RapidScan™ Gene Expression Panel was used to generate acomprehensive expression profile of the putative GPCR in human tissues.Human tissues in the array may include: brain, heart, kidney, spleen,liver, colon, lung, small intestine, muscle, stomach, testis, placenta,salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus,prostate, skin, PBL, bone marrow, fetal brain, fetal liver. Human brainregions in the array may include: frontal lobe, temporal lobe,cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala,thalamus, hypothalamus, pons, medulla and spinal cord.

Expression of the nGPCR-1 in the various tissues was detected by usingPCR primers designed based on the available sequence of the receptorthat will prime the synthesis of a 212 bp fragment in the presence ofthe appropriate cDNA. The forward primer was:

GCTCAACCCACTCATCTATGCC (SEQ ID NO: 97), and the reverse primer was:

AAACTTCTCTGCCCTTACCGTC (SEQ ID NO: 98)

The PCR reaction mixture was added to each well of the PCR plate. Theplate was placed in a GeneAmp PCR9700 PCR thermocycler (Perkin ElmerApplied Biosystems). The plate was then exposed to the following cyclingparameters: Pre-soak 94° C. for 3 min; denaturation at 94° C. for 30seconds; annealing at primer T_(m) for 45 seconds; extension 72° C. for2 minutes; for 35 cycles. PCR products were then separated and analyzedby electrophoresis on a 1.5-% agarose gel.

The 4-log dilution range of cDNA deposited on the plate ensured that theamplification reaction is within the linear range and, hence,facilitated the semi-quantitative determination of relative mRNAaccumulation in the various tissues or brain regions examined.

Expression of nGPCR-1 was found to be highest in the testis, adrenalgland and heart. Significant levels of expression were also found in thebrain, kidney, spleen ovary, prostate, muscle, PBL, stomach and bonemarrow. Within the brain, expression levels were highest in thecerebellum, amygdala, thalamus and spinal cord, with significant levelsof expression in the frontal lobe, hippocampus, substantia nigra,hypothalamus and pons.

Expression of nGPCR-1 in the brain provided an indication thatmodulators of nGPCR-1 activity have utility for treating neurologicaldisorders, including but not limited to, schizophrenia, affectivedisorders, ADHD/ADD (i.e., Attention Deficit-HyperactivityDisorder/Attention Deficit Disorder), and neural disorders such asAlzheimer's disease, Parkinson's disease, migraine, and senile dementia.Some other diseases for which modulators of nGPCR-1 may have utilityinclude depression, anxiety, bipolar disease, epilepsy, neuritis,neurasthenia, neuropathy, neuroses, and the like. Use of nGPCR-1modulators, including nGPCR-1 ligands and ant-nGPCR-1 antibodies, totreat individuals having such disease states is intended as an aspect ofthe invention.

nGPCR-3

Tissue specific expression of the cDNA encoding nGPCR-3 was detectedusing a PCR-based method. Multiple Choice™ first strand cDNAs (OriGeneTechnologies, Rockville, Md.) from 6 human tissues were serially dilutedover a 3-log range and arrayed into a multi-well PCR plate. This arraywas used to generate a comprehensive expression profile of the putativeGPCR in human tissues. Human tissues arrayed included: brain, heart,kidney, peripheral blood leukocytes, lung and testis. PCR primers weredesigned based on the available sequence of the putative GPCR. Thesequence of the forward primer used was:

5′TGCTGCTTTGTTGCGCCTAC3′ (SEQ ID NO: 189), corresponding to base pairs77 through 96 of the predicted coding sequence of nGPCR-3. The sequenceof the reverse primer used was:

5′TTGGACGCCAGGAAGGTG3′ (SEQ ID NO: 190), corresponding to base pairs 258through 285 of the predicted coding sequence of nGPCR-3. This primer setprimes the synthesis of a 298 base pair fragment in the presence of theappropriate cDNA. For detection of expression within brain regions, thesame primer set was used with the Human Brain Rapid Scan™ Panel (OriGeneTechnologies, Rockville, Md.). This panel represents serial dilutionsover a 3 log range of first strand cDNA from the following brain regionsarrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum,hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus,hypothalamus, pons, medulla and spinal cord. Primers were synthesized byGenosys Corp., The Woodlands, Tex. PCR reactions were assembled usingthe components of the Expand Hi-Fi PCR System (Roche MolecularBiochemicals, Indianapolis, Ind.). Twenty-five microliters of the PCRreaction mixture was added to each well of the RapidScan PCR plate. Theplate was placed in a GeneAmp 9700 PCR thermocycler (Perkin ElmerApplied Biosystems). The following cycling program was executed:Pre-soak at (94° C. for 3 min.) followed by 35 cycles of [(94° C. for 45sec.), (53° C. for 2 min.), and (72° C. for 45 sec.)]. PCR reactionproducts were then separated and analyzed by electrophoresis on a 2.0%agarose gel stained with ethidium bromide.

The results indicated that nGPCR-3 was expressed in the brain, heart,kidney, peripheral blood lymphocytes, lung, and testis. In the brain,nGPCR-3 was expressed in frontal lobe, temporal lobe, cerebellum,hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus,hypothalamus, pons, medulla, as well as in the spinal cord.

nGPCR-9

The RapidScan™ Gene Expression Panel was used to generate acomprehensive expression profile of the putative GPCR in human tissues.Human tissues arrayed include: brain, heart, kidney, spleen, liver,colon, lung, small intestine, muscle, stomach, testis, placenta,salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus,prostate, skin, PBL, bone marrow, fetal brain, fetal liver.

The forward primer used was to detect expression of nGPCR-9 was:

5′ AACCCCATCATCTACACGC 3′(SEQ ID NO: 105), and, the reverse primer was:

5′ TGCCTGTGGAGCCGCTGG 3′(SEQ ID NO: 106). This primer set will prime thesynthesis of a 238 base pair fragment in the presence of the appropriatecDNA.

For detection of expression within brain regions, the same primer setwas used with the Human Brain Rapid Scan™ Panel (OriGene Technologies,Rockville, Md.). This panel represents serial dilutions over a 2-logrange of first strand cDNA from the following brain regions arrayed in a96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus,substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus,pons, medulla and spinal cord.

Twenty-five microliters of the PCR reaction mixture was added to eachwell of the PCR plate. The plate was placed in a GeneAmp 9700 PCRthermocycler (Perkin Elmer Applied Biosystems). The following cyclingprogram was executed: Pre-soak at (94° C. for 3 min.) followed by 35cycles of [(94° C. for 45 sec.) (52° C. for 2 min.) (72° C. for 45sec.)]. PCR reaction products were then separated and analyzed byelectrophoresis on a 2.0% agarose gel and stained with ethidium bromide.

nGPCR-9 was expressed in the brain, peripheral blood leukocytes, heart,kidney, adrenal gland, spleen, pancreas, liver, lung, skin, bone marrow,testis, placenta, salivary gland, uterus, small intestine, muscle,stomach, and fetal liver. Within the brain, nGPCR-9 was expressed in allareas examined including the frontal lobe, temporal lobe, cerebellum,hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus,hypothalamus, pons, medulla and spinal cord.

Expression of nGPCR-9 in the brain provided an indication thatmodulators of nGPCR-9 activity have utility for treating disorders,including but not limited to, schizophrenia, affective disorders,movement disorders, metabolic disorders, inflammatory disorders,cancers, ADHD/ADD (i.e., Attention Deficit-HyperactivityDisorder/Attention Deficit Disorder), and neural disorders such asAlzheimer's disease, Parkinson's disease, migraine, and senile dementia.Use of nGPCR-9 modulators, including nGPCR-9 ligands and anti-nGPCR-9antibodies, to treat individuals having such disease states is intendedas an aspect of the invention.

nGPCR-41

The RapidScan™ Gene Expression Panel was used to generate acomprehensive expression profile of the putative GPCR in human tissues.Human tissues in the array included, inter alia: brain, heart, kidney,spleen, liver, colon, lung, small intestine, muscle, stomach, testis,placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary,uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.Human brain regions in the array included, inter alia: frontal lobe,temporal lobe, cerebellum, hippocampus, substantia nigra, caudatenucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinalcord.

Expression of nGPCR-11 in the various tissues was detected by using PCRprimers designed based on the available sequence of the receptor thatwill prime the synthesis of a 206 bp fragment in the presence of theappropriate cDNA. The forward primer used to detect expression ofnGPCR-11 was:

5′-GAAGCCCAGCACTGTTTACC-3′ (SEQ ID NO: 109), and the reverse primer was:

5′-TGAAATACCTGTCCGCAGCC-3 (SEQ ID NO: 10).

Twenty-five microliters of the PCR reaction mixture was added to eachwell of the RapidScan PCR plate. The plate was placed in a GeneAmp 9700PCR thermocycler (PE Applied Biosystems). The following cycling programwas executed: Pre-soak 94° C. for 3 min; denaturation at 94° C. for 30seconds; annealing at primer T_(m) for 45 seconds; extension at 72° C.for 2 minutes; for 35 cycles. PCR reaction products were then separatedand analyzed by electrophoresis on a 2.0% agarose gel stained withethidium bromide.

The 4-log dilution range of cDNA deposited on the plate ensured that theamplification reaction was within the linear range and, facilitatedsemi-quantitative determination of relative mRNA accumulation in thevarious tissues or brain regions examined.

nGPCR-11 was expressed in the thyroid gland, brain, heart, kidney,adrenal gland, spleen, liver, ovary, muscle, testis, salivary gland,colon, prostate, small intestine, skin stomach, bone marrow, fetal brainand placenta. Within the brain, nGPCR-11 was expressed in the temporallobe, amygdala, substantia nigra, pons, spinal cord, frontal lobe, andcerebellum.

Expression of the nGPCR-11 in the brain provided an indication thatmodulators of nGPCR-11 activity have utility for treating disorders,including but not limited to, schizophrenia, affective disorders,metabolic disorders, inflammatory disorders, cancers, ADHD/ADD (i.e.,Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder),and neural disorders such as Alzheimer's disease, Parkinson's disease,migraine, and senile dementia. Some other diseases for which modulatorsof nGPCR-11 may have utility include depression, anxiety, bipolardisease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and thelice. Use of nGPCR-11 modulators, including nGPCR1-11 ligands andanti-nGPCR-11 antibodies, to treat individuals having such diseasestates is intended as an aspect of the invention.

Expression of nGPCR-11 in the thyroid gland, indicates that agonists orantagonists could be of use in the treatment of thyroid dysfunction suchas thyreotoxicosis and myxoedema. They could also be of use in thestimulation of thyroid hormone release leading to overall increase inmetabolic rate and weight reduction. The expression of nGPCR-11 in liverand muscle indicate a use for agonists or antagonists in regulation ofglucose metabolism applicable in diabetes type II.

nGCPR-16

The RapidScan™ Gene Expression Panel was used to generate acomprehensive expression profile of the putative GPCR in human tissues.Human tissues in the array included, inter alia: brain, heart, kidney,spleen, liver, colon, lung, small intestine, muscle, stomach, testis,placenta, salivary gland, thyroid, adrenal gland, pancreas, ovary,uterus, prostate, skin, PBL, bone marrow, fetal brain, fetal liver.Human brain regions in the array included, inter alia: frontal lobe,temporal lobe, cerebellum, hippocampus, substantia nigra, caudatenucleus, amygdala, thalamus, hypothalamus, pons, medulla and spinalcord.

Expression of nGPCR-16 in the various tissues was detected by using PCRprimers designed based on the available sequence of the receptor thatwill prime the synthesis of a 205 bp fragment in the presence of theappropriate cDNA. The forward primer used to detect expression ofnGPCR-16 was:

5′ CAGCCCAAACATCCAAGTC 3′. (SEQ ID NO: 113). The reverse primer used todetect expression of nGPCR-16 was:

5′ ACCCCACTTAATCAGCCTC 3′ (SEQ ID NO: 114).

For detection of expression within brain regions, the same primer setwas used with the Human Brain Rapid Scan™ Panel (OriGene Technologies,Rockville, Md.). This panel represents serial dilutions over a 2 logrange of first strand cDNA from the following brain regions arrayed in a96 well format: frontal lobe, temporal lobe, cerebellum, hippocampus,substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus,pons, medulla and spinal cord.

Twenty-five microliters of the PCR reaction mixture was added to eachwell of the RapidScan PCR plate. The plate was placed in a GeneAmp 9700PCR thermocycler (Perkin Elmer Applied Biosystems). The followingcycling program was executed: Pre-soak at (94° for 3 min.) followed by35 cycles of [(94° C. for 45 sec.) (53° C. for 2 min.) (72° C. for 45sec.)]. PCR reaction products were then separated and analyzed byelectrophoresis on a 2.0% agarose gel, and stained with ethidiumbromide.

The 4-log dilution range of cDNA deposited on the plate ensured that theamplification reaction was within the linear range and, facilitatedsemi-quantitative determination of relative mRNA accumulation in thevarious tissues or brain regions examined.

nGPCR-16 was expressed in the ovary, lung, prostate, bone marrow,salivary gland, heart, adrenal gland, spleen, liver, small intestine,skin, muscle, peripheral blood leukocytes, testis, placenta, fetalliver, brain, thyroid gland, kidney, pancreas, colon, uterus, andstomach. Within the brain, nGPCR-16 was expressed in all areas examinedincluding the frontal lobe, temporal lobe, cerebellum, hippocampus,substantia nigra, caudate nucleus, amygdala, thalamus, hypothalamus,pons, medulla and spinal cord.

Expression of nGPCR-16 in the brain provides an indication thatmodulators of nGPCR-16 activity have utility for treating neurologicaldisorders, including but not limited to, schizophrenia, affectivedisorders, ADHD/ADD (i.e., Attention Deficit-HyperactivityDisorder/Attention Deficit Disorder), and neural disorders such asAlzheimer's disease, Parkinson's disease, migraine, and senile dementia.Some other diseases for which modulators of nGPCR-16 may have utilityinclude depression, anxiety, bipolar disease, epilepsy, neuritis,neurasthenia, neuropathy, neuroses, and the like. Use of nGPCR-16modulators, including nGPCR-16 ligands and anti-nGPCR-16 antibodies, totreat individuals having such disease states is intended as an aspect ofthe invention.

nGPCR-40

The RapidScan™ Gene Expression Panel (OriGene Technologies, Rockville,Md.) was used to generate a comprehensive expression profile of theputative GPCR in human tissues. Human tissues arrayed include: brain,heart, kidney, spleen, liver, colon, lung, small intestine, muscle,stomach, testis, placenta, salivary gland, thyroid, adrenal gland,pancreas, ovary, uterus, prostate, skin, PBL, bone marrow, fetal brain,fetal liver. The forward primer used was:

5′ACAGCCCCAAAGCCAAACAC3′, (SEQ ID NO: 117), and the reverse primer was:

5′CCGCAGGAGCAATGAAAATCAG3′, (SEQ ID NO: 118). This primer set primed thesynthesis of a 220 base pair fragment in the presence of the appropriatecDNA. For detection of expression within brain regions, the same primerset was used with the Human Brain RapidScan™ Panel (OriGeneTechnologies, Rockville, Md.). This panel represents serial dilutionsover a 2 log range of first strand cDNA from the following brain regionsarrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum,hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus,hypothalamus, pons, medulla and spinal cord.

Twenty-five microliters of the PCR reaction mixture was added to eachwell of the RapidScan PCR plate. The plate was placed in a GeneAmp 9700PCR thermocycler (Perkin Elmer Applied Biosystems). The followingcycling program was executed: Pre-soak at (94° C. for 3 min.) followedby 35 cycles of [(94° for 45 sec.) (54° C. for 2 min.) (72° for 45sec.)]. PCR reaction products were then separated and analyzed byelectrophoresis on a 2.0% agarose gel stained with ethidium bromide.

The dilution range of cDNA deposited on the plates ensured that theamplification reaction was within the linear range and, hence,facilitated semi-quantitative determination of relative mRNAaccumulation in the various tissues or brain regions examined.

nGPCR-40 was expressed in the brain, peripheral blood lymphocytes,pancreas, ovary, uterus, testis, salivary gland, kidney, adrenal gland,liver, bone marrow, prostate, fetal liver, colon, muscle, and fetalbrain, may be found in many other tissues, including, but not limitedto, lung, small intestine, fetal brain cord, and bone. Within the brain,nGPCR-40 was expressed in the frontal lobe, hypothalamus, pons,cerebellum, caudate nucleus, and medulla.

Expression of nGPCR-40 in the brain provides an indication thatmodulators of nGPCR-40 activity have utility for treating neurologicaldisorders, including but not limited to, movement disorders, affectivedisorders, metabolic disorders, inflammatory disorders and cancers. Useof nGPCR-40 modulators, including nGPCR-40 ligands and anti-nGPCR-40antibodies, to treat individuals having such disease states is intendedas an aspect of the invention.

nGPCR-54

Multiple Choice™ first strand cDNAs (OriGene Technologies, Rockville,Md.) from 12 human tissues were serially diluted over a 3-log range andarrayed into a multi-well PCR plate. Human tissues arrayed include:brain, heart, kidney, peripheral blood leukocytes, liver, lung, muscle,ovary, prostate, small intestine, spleen and testis. PCR primers weredesigned based on the sequence of nGPCR-54 provided herein. The forwardprimer used was:

5′CTGTCTCTCTGTCCTCTTCC3′, (SEQ ID NO: 123). The reverse primer used was:

5′GCACCGATCTTCATTGAATITC3′, (SEQ ID NO: 124). This primer set primes thesynthesis of a 145 base pair fragment in the presence of the appropriatecDNA. For detection of expression within brain regions, the same primerset was used with the Human Brain Rapid Scan™ Panel (OriGeneTechnologies, Rockville, Md.). This panel represents serial dilutionsover a 3 log range of first strand cDNA from the following brain regionsarrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum,hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus,hypothalamus, pons, medulla and spinal cord.

Twenty-five microliters of the PCR reaction mixture was added to eachwell of the RapidScan PCR plate. The plate was placed in a GeneAmp 9700PCR thermocycler (Perkin Elmer Applied Biosystems). The followingcycling program was executed: Pre-soak at (94° C. for 3 min.) followedby 35 cycles of [(94° C. for 45 sec.) (52.5° C. for 2 min.) (72° C. for45 sec.)]. PCR reaction products were then separated and analyzed byelectrophoresis on a 2.0% agarose gel stained with ethidium bromide.

nGPCR-54 was expressed in the brain, kidney, lung, muscle, testis,heart, liver, ovary, prostate, small intestine, spleen, and peripheralblood leukocytes. Within the brain, nGPCR-54 was expressed in thecerebellum, hippocampus, substantia nigra, thalamus, hypothalamus, pons,frontal lobe, temporal lobe, caudate nucleus, medulla, spinal cord, andamygdala.

Expression of the nGPCR-54 in the brain provides an indication thatmodulators of nGPCR-54 activity have utility for treating neurologicaldisorders, including but not limited to, movement disorders, affectivedisorders, metabolic disorders, inflammatory disorders and cancers. Useof nGPCR-54 modulators, including nGPCR-54 ligands and anti-nGPCR-54antibodies, to treat individuals having such disease states is intendedas an aspect of the invention.

nGPCR-56

The RapidScan™ Gene Expression Panel was used to generate acomprehensive expression profile of the putative GPCR in human tissues.Human tissues arrayed include: brain, heart, kidney, spleen, liver,colon, lung, small intestine, muscle, stomach, testis, placenta,salivary gland, thyroid, adrenal gland, pancreas, ovary, uterus,prostate, skin, PBL, bone marrow, fetal brain, fetal liver. The forwardprimer used was:

5′ ACTRCAAACAACTTCATACCCC 3′ (SEQ ID NO: 125), and the reverse primerused was:

5′ACACACAGCATAGTAGCG 3′ (SEQ ID NO: 126). This primer set will prime thesynthesis of a 231 base pair fragment in the presence of the appropriatecDNA. For detection of expression within brain regions, the same primerset was used with the Human Brain Rapid Scan™ Panel (OriGeneTechnologies, Rockville, Md.). This panel represents serial dilutionsover a 2 log range of first strand cDNA from the following brain regionsarrayed in a 96 well format: frontal lobe, temporal lobe, cerebellum,hippocampus, substantia nigra, caudate nucleus, amygdala, thalamus,hypothalamus, pons, medulla and spinal cord.

Twenty-five microliters of the PCR reaction mixture was added to eachwell of the RapidScan PCR plate. The plate was placed in a GeneAmp 9700PCR thermocycler (Perkin Elmer Applied Biosystems). The followingcycling program was executed: Pre-soak at (94° C. for 3 min.) followedby 35 cycles of [(94° C. for 45 sec.) (53° C. for 2 min.) (72° C. for 45sec.)]. PCR reaction products were then separated and analyzed byelectrophoresis on a 2.0% agarose gel stained with ethidium bromide.

nGPCR-56 was expressed in peripheral blood lymphocytes, testis, salivarygland, kidney, spleen, skin, stomach, placenta, ovary, bone marrow,fetal liver, small intestine, and fetal brain.

Expression of nGPCR-56 in the brain provides an indication thatmodulators of nGPCR-56 activity have utility for treating neurologicaldisorders, including but not limited to, movement disorders, affectivedisorders, metabolic disorders, inflammatory disorders and cancers. Useof nGPCR-56 modulators, including nGPCR-56 ligands and anti-nGPCR-56antibodies, to treat individuals having such disease states is intendedas an aspect of the invention.

nGPCR-58

The RapidScan™ Gene Expression Panel was used to generate acomprehensive expression profile of the putative GPCR in human tissues.Human tissues in the array included: brain, heart, kidney, spleen,liver, lung, small intestine, muscle, testis, ovary, prostate, and PBL.Human brain regions in the array included: frontal lobe, temporal lobe,cerebellum, hippocampus, substantia nigra, caudate nucleus, amygdala,thalamus, hypothalamus, pons, medulla and spinal cord.

Expression of the nGPCR-58 in the various tissues was detected by usingPCR primers designed based on the available sequence of the receptorthat will prime the synthesis of a 282 bp fragment in the presence ofthe appropriate cDNA. The forward primer was:

CAGAGCTTGATGATGAGGAC (SEQ ID NO: 127), and the reverse primer was:

CCCATAGGAAGTAGTAGAAG (SEQ ID NO: 128).

The PCR reaction mixture was added to each well of the PCR plate. Theplate was placed in a GeneAmp PCR9700 PCR thermocycler (Perkin ElmerApplied Biosystems). The plate was then exposed to the following cyclingparameters: Pre-soak 94° for 3 min; denaturation at 94° for 30 seconds;annealing at primer T_(m) for 45 seconds; extension at 72° for 2minutes; for 35 cycles. PCR productions were then separated and analyzedby electrophoresis on a 1.5-% agarose gel.

The 4-log dilution range of cDNA deposited on the plate ensured that theamplification reaction was within the linear range and, hence,facilitated semi-quantitative determination of relative mRNAaccumulation in the various tissues or brain regions examined.

nGPCR-58 was expressed in all tissues included on the array, includingbrain, muscle, prostate, kidney, peripheral blood lymphocytes, liver,lung, small intestine, spleen, testis, heart, and ovary. Within thebrain, nGPCR-58 was expressed in many regions including, but not limitedto cerebellum, substantia nigra, thalamus, pons, spinal cord, frontallobe, temporal lobe, hippocampus, caudate nucleus, amygdala,hypothalamus, and medulla.

Expression of the nGPCR-58 in the brain provided an indication thatmodulators of nGPCR-58 activity have utility for treating disorders,including but not limited to, schizophrenia, affective disorders,ADHD/ADD (i.e., Attention Deficit-Hyperactivity Disorder/AttentionDeficit Disorder), neural disorders such as Alzheimer's disease,Parkinson's disease, migraine, senile dementia, depression, anxiety,bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses,metabolic disorders, inflammatory disorders, cancers and the like. Useof nGPCR-58 modulators, including nGPCR-58 ligands and anti-nGPCR-58antibodies, to treat individuals having such disease states is intendedas an aspect of the invention.

Example 5 Northern Blot Analysis

Northern blots are performed to examine the expression of nGPCR-x mRNA.The sense orientation oligonucleotide and the antisense-orientationoligonucleotide, described above, are used as primers to amplify aportion of the GPCR-x cDNA sequence of an odd numbered nucleotidesequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 andSEQ ID NO:191.

Multiple human tissue northern blots from Clontech (Human II # 7767-1)are hybridized with the probe. Pre-hybridization is carried out at 42 Cfor 4 hours in 5×SSC, 1× Denhardt's reagent, 0.1% SDS, 50% formamide,250 mg/ml salmon sperm DNA. Hybridization is performed overnight at 42°C. in the same mixture with the addition of about 1.5×10⁶ cpm/ml oflabeled probe.

The probe is labeled with α-³²P-DCTP by Rediprime™ DNA labeling system(Amersham Pharmacia), purified on Nick Column (Amersham Pharmacia) andadded to the hybridization solution. The filters are washed severaltimes at 42 C in 0.2×SSC, 0.1% SDS. Filters are exposed to Kodak XARfilm (Eastman Kodak Company, Rochester, N.Y., USA) with intensifyingscreen at −80° C.

Example 6 Recombinant Expression of nGPCR-x in Eukaryotic Host Cells

A. Expression of nGPCR-x in Mammalian Cells

To produce nGPCR-x protein, a nGPCR-x-encoding polynucleotide isexpressed in a suitable host cell using a suitable expression vector andstandard genetic engineering techniques. For example, thenGPCR-x-encoding sequence described in Example 1 is subcloned into thecommercial expression vector pzeoSV2 (Invitrogen, San Diego, Calif.) andtransfected into Chinese Hamster Ovary (CHO) cells using thetransfection reagent FuGENE6™ (Boehringer-Mannheim) and the transfectionprotocol provided in the product insert. Other eukaryotic cell lines,including human embryonic kidney (HEK 293) and COS cells, are suitableas well. Cells stably expressing nGPCR-x are selected by growth in thepresence of 100 μg/ml zeocin (Stratagene, LaJolla, Calif.). Optionally,nGPCR-x may be purified from the cells using standard chromatographictechniques. To facilitate purification, antisera is raised against oneor more synthetic peptide sequences that correspond to portions of thenGPCR-x amino acid sequence, and the antisera is used to affinity purifynGPCR-x. The nGPCR-x also may be expressed in-frame with a tag sequence(e.g. polyhistidine, hemagluttinin, FLAG) to facilitate purification.Moreover, it will be appreciated that many of the uses for nGPCR-xpolypeptides, such as assays described below, do not requirepurification of nGPCR-x from the host cell.

B. Expression of nGPCR-x in 293 Cells

For expression of nGPCR-x in mammalian cells 293 (transformed human,primary embryonic kidney cells), a plasmid bearing the relevant nGPCR-xcoding sequence is prepared, using vector pSecTag2A (Invitrogen). VectorpSecTag2A contains the murine IgK chain leader sequence for secretion,the c-myc epitope for detection of the recombinant protein with theanti-myc antibody, a C-terminal polyhistidine for purification withnickel chelate chromatography, and a Zeocin resistant gene for selectionof stable transfectants. The forward primer for amplification of thisGPCR cDNA is determined by routine procedures and preferably contains a5′ extension of nucleotides to introduce the HindIII cloning site andnucleotides matching the GPCR sequence. The reverse primer is alsodetermined by routine procedures and preferably contains a 5′ extensionof nucleotides to introduce an XhoI restriction site for cloning andnucleotides corresponding to the reverse complement of the nGPCR-xsequence. The PCR conditions are 55° C. as the annealing temperature.The PCR product is gel purified and cloned into the HindIII-XhoI sitesof the vector.

The DNA is purified using Qiagen chromatography columns and transfectedinto 293 cells using DOTAP™ transfection media (Boehringer Mannheim,Indianapolis, Ind.). Transiently transfected cells are tested forexpression after 24 hours of transfection, using western blots probedwith anti-His and anti-nGPCR-x peptide antibodies. Permanentlytransfected cells are selected with Zeocin and propagated. Production ofthe recombinant protein is detected from both cells and media by westernblots probed with anti-His, anti-Myc or anti-GPCR peptide antibodies.

C. Expression of nGPCR-x in COS Cells

For expression of the nGPCR-x in COS7 cells, a polynucleotide moleculehaving an odd numbered nucleotide sequence ranging from SEQ ID NO: 1 toSEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can be cloned intovector p3-CI. This vector is a pUC18-derived plasmid that contains theHCMV (human cytomegalovirus) promoter-intron located upstream from thebGH (bovine growth hormone) polyadenylation sequence and a multiplecloning site. In addition, the plasmid contains the dhrf (dihydrofolatereductase) gene which provides selection in the presence of the drugmethotrexane (WIX) for selection of stable transformants.

The forward primer is determined by routine procedures and preferablycontains a 5′ extension which introduces an XbaI restriction site forcloning, followed by nucleotides which correspond to an odd numberednucleotide sequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ IDNO: 185 and SEQ ID NO:191. The reverse primer is also determined byroutine procedures and preferably contains 5′-extension of nucleotideswhich introduces a SalI cloning site followed by nucleotides whichcorrespond to the reverse complement of an odd numbered nucleotidesequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 andSEQ ID NO:191. The PCR consists of an initial denaturation step of 5 minat 95° C. 30 cycles of 30 sec denaturation at 95° C., 30 sec annealingat 58° C. and 30 sec extension at 72° C., followed by 5 min extension at72° C. The PCR product is gel purified and ligated into the XbaI andSalI sites of vector p3-CI. This construct is transformed into E. colicells for amplification and DNA purification. The DNA is purified withQiagen chromatography columns and transfected into COS 7 cells usingLipofectamine™ reagent from BRL, following the manufacturer's protocols.Forty-eight and 72 hours after transfection, the media and the cells aretested for recombinant protein expression.

nGPCR-x expressed from a COS cell culture can be purified byconcentrating the cell-growth media to about 10 mg of protein/ml, andpurifying the protein by, for example, chromatography. Purified nGPCR-xis concentrated to 0.5 mg/ml in an Amicon concentrator fitted with aYM-10 membrane and stored at −80° C.

D. Expression of nGPCR-x in Insect Cells

For expression of nGPCR-x in a baculovirus system, a polynucleotidemolecule having an odd numbered nucleotide sequence ranging from SEQ IDNO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191 can beamplified by PCR. The forward primer is determined by routine proceduresand preferably contains a 5′ extension which adds the NdeI cloning site,followed by nucleotides which correspond to an odd numbered nucleotidesequence ranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 andSEQ ID NO:191. The reverse primer is also determined by routineprocedures and preferably contains a 5′ extension which introduces theKpnI cloning site, followed by nucleotides which correspond to thereverse complement of an odd numbered nucleotide sequence ranging fromSEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ ID NO:191.

The PCR product is gel purified, digested with NdeI and KpnI, and clonedinto the corresponding sites of vector pACHTL-A (Pharmingen, San Diego,Calif.). The pAcHTL expression vector contains the strong polyhedrinpromoter of the Autographa californica nuclear polyhedrosis virus(AcMNPV), and a 6×His tag upstream from the multiple cloning site. Aprotein kinase site for phosphorylation and a thrombin site for excisionof the recombinant protein precede the multiple cloning site is alsopresent. Of course, many other baculovirus vectors could be used inplace of pAcHTL-A, such as pAc373, pVL941 and pAcIM1. Other suitablevectors for the expression of GPCR polypeptides can be used, providedthat the vector construct includes appropriately located signals fortranscription, translation, and trafficking, such as an in-frame AUG anda signal peptide, as required. Such vectors are described in Luckow etal., Virology 170:31-39, among others.

The virus is grown and isolated using standard baculovirus expressionmethods, such as those described in Summers et al. (A Manual of Methodsfor Baculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987)).

In a preferred embodiment, pAcHLT-A containing nGPCR-x gene isintroduced into baculovirus using the “BaculoGold™” transfection kit(Pharmingen, San Diego, Calif.) using methods established by themanufacturer. Individual virus isolates are analyzed for proteinproduction by radiolabeling infected cells with ³⁵S-methionine at 24hours post infection. Infected cells are harvested at 48 hours postinfection, and the labeled proteins are visualized by SDS-PAGE. Virusesexhibiting high expression levels can be isolated and used for scaled upexpression.

For expression of a nGPCR-x polypeptide in a Sf9 cells, a polynucleotidemolecule having the sequence of an odd numbered nucleotide sequenceranging from SEQ ID NO: 1 to SEQ ID NO: 93, SEQ ID NO: 185 and SEQ IDNO:191 can be amplified by PCR using the primers and methods describedabove for baculovirus expression. The nGPCR-x cDNA is cloned into vectorpAcHLT-A (Pharmingen) for expression in Sf9 insect. The insert is clonedinto the NdeI and KpnI sites, after elimination of an internal NdeI site(using the same primers described above for expression in baculovirus).DNA is purified with Qiagen chromatography columns and expressed in Sf9cells. Preliminary Western blot experiments from non-purified plaquesare tested for the presence of the recombinant protein of the expectedsize which reacted with the GPCR-specific antibody. These results areconfirmed after further purification and expression optimization in HiG5cells.

Example 7 Interaction Trap/Two-Hybrid System

In order to assay for nGPCR-x-interacting proteins, the interactiontrap/two-hybrid library screening method can be used. This assay wasfirst described in Fields et al., Nature, 1989, 340, 245, which isincorporated herein by reference in its entirety. A protocol ispublished in Current Protocols in Molecular Biology 1999, John Wiley &Sons, NY, and Ausubel, F. M et al. 1992, Short protocols in molecularbiology, Fourth edition, Greene and Wiley-interscience, NY, each ofwhich is incorporated herein by reference in its entirety. Kits areavailable from Clontech, Palo Alto, Calif. (Matchmaker Two-Hybrid System3).

A fusion of the nucleotide sequences encoding all or partial nGPCR-x andthe yeast transcription factor GAL4 DNA-binding domain (DNA-BD) isconstructed in an appropriate plasmid (ie., pGBKT7) using standardsubcloning techniques. Similarly, a GALA active domain (AD) fusionlibrary is constructed in a second plasmid (i.e., pGADT7) from cDNA ofpotential GPCR-binding proteins (for protocols on forming cDNAlibraries, see Sambrook et al. 1989, Molecular cloning: a laboratorymanual, second edition, Cold Spring Harbor Press, Cold Spring Harbor,N.Y.), which is incorporated herein by reference in its entirety. TheDNA-BD/nGPCR-x fusion construct is verified by sequencing, and testedfor autonomous reporter gene activation and cell toxicity, both of whichwould prevent a successful two-hybrid analysis. Similar controls areperformed with the AD/library fusion construct to ensure expression inhost cells and lack of transcriptional activity. Yeast cells aretransformed (ca. 105 transformants/mg DNA) with both the nGPCR-x andlibrary fusion plasmids according to standard procedures (Ausubel etal., 1992, Short protocols in molecular biology, fourth edition, Greeneand Wiley-interscience, NY, which is incorporated herein by reference inits entirety). In vivo binding of DNA-BD/nGPCR-x with AD/libraryproteins results in transcription of specific yeast plasmid reportergenes (i.e., lacZ, HIS3, ADE2, LEU2). Yeast cells are plated onnutrient-deficient media to screen for expression of reporter genes.Colonies are dually assayed for β-galactosidase activity upon growth inXgal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) supplemented media(filter assay for β-galactosidase activity is described in Breeden etal., Cold Spring Harb. Symp. Quant. Biol., 1985, 50, 643, which isincorporated herein by reference in its entirety). Positive AD-libraryplasmids are rescued from transformants and reintroduced into theoriginal yeast strain as well as other strains containing unrelatedDNA-BD fusion proteins to confirm specific nGPCR-x/library proteininteractions. Insert DNA is sequenced to verify the presence of an openreading frame fused to GAL4 AD and to determine the identity of thenGPCR-x-binding protein.

Example 8 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis

A gel electrophoresis mobility shift assay can rapidly detect specificprotein-DNA interactions. Protocols are widely available in such manualsas Sambrook et al. 1989, Molecular cloning: a laboratory manual, secondedition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. and Ausubel,F. M. et al., 1992, Short Protocols in Molecular Biology, fourthedition, Greene and Wiley-interscience, NY, each of which isincorporated herein by reference in its entirety.

Probe DNA (<300 bp) is obtained from synthetic oligonucleotides,restriction endonuclease fragments, or PCR fragments and end-labeledwith ³²P. An aliquot of purified nGPCR-x (ca. 15 μg) or crude nGPCR-xextract (ca. 15 ng) is incubated at constant temperature (in the range22-37 C) for at least 30 minutes in 10-15 μl of buffer (i.e. TAE or TBE,pH 8.0-8.5) containing radiolabeled probe DNA, nonspecific carrier DNA(ca 1 μg), BSA (300 μg/ml), and 10% (v/v) glycerol. The reaction mixtureis then loaded onto a polyacrylamide gel and run at 30-35 mA until goodseparation of free probe DNA from protein-DNA complexes occurs. The gelis then dried and bands corresponding to free DNA and protein-DNAcomplexes are detected by autoradiography.

Example 9 Antibodies to nGPCR-X

Standard techniques are employed to generate polyclonal or monoclonalantibodies to the nGPCR-x receptor, and to generate usefulantigen-binding fragments thereof or variants thereof, including“humanized” variants. Such protocols can be found, for example, inSambrook et al. (1989) and Harlow et al. (Eds.), Antibodies A LaboratoryManual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988).In one embodiment, recombinant nGPCR-x polypeptides (or cells or cellmembranes containing such polypeptides) are used as antigen to generatethe antibodies. In another embodiment, one or more peptides having aminoacid sequences corresponding to an immunogenic portion of nGPCR-x (e.g.,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more aminoacids) are used as antigen. Peptides corresponding to extracellularportions of nGPCR-x, especially hydrophilic extracellular portions, arepreferred. The antigen may be mixed with an adjuvant or linked to ahapten to increase antibody production.

A. Polyclonal or Monoclonal Antibodies

As one exemplary protocol, recombinant nGPCR-x or a synthetic fragmentthereof is used to immunize a mouse for generation of monoclonalantibodies (or larger mammal, such as a rabbit, for polyclonalantibodies). To increase antigenicity, peptides are conjugated toKeyhole Lympet Hemocyanin (Pierce), according to the manufacturer'srecommendations. For an initial injection, the antigen is emulsifiedwith Freund's Complete Adjuvant and injected subcutaneously. Atintervals of two to three weeks, additional aliquots of nGPCR-x antigenare emulsified with Freund's Incomplete Adjuvant and injectedsubcutaneously. Prior to the final booster injection, a serum sample istaken from the immunized mice and assayed by western blot to confirm thepresence of antibodies that immunoreact with nGPCR-x. Serum from theimmunized animals may be used as polyclonal antisera or used to isolatepolyclonal antibodies that recognize nGPCR-x. Alternatively, the miceare sacrificed and their spleen removed for generation of monoclonalantibodies.

To generate monoclonal antibodies, the spleens are placed in 10 mlserum-free RPMI 1640, and single cell suspensions are formed by grindingthe spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine,1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/mlstreptomycin (RPMI) (Gibco, Canada). The cell suspensions are filteredand washed by centrifugation and resuspended in serum-free RPMI.Thymocytes taken from three naive Balb/c mice are prepared in a similarmanner and used as a Feeder Layer. NS-1 myeloma cells, kept in log phasein RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc.,Logan, Utah) for three days prior to fusion, are centrifuged and washedas well.

To produce hybridoma fusions, spleen cells from the immunized mice arecombined with NS-1 cells and centrifuged, and the supernatant isaspirated. The cell pellet is dislodged by tapping the tube, and 2 ml of37° C. PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer-Mannheim) isstirred into the pellet, followed by the addition of serum-free RPMI.Thereafter, the cells are centrifuged, resuspended in RPMI containing15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine(HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×10⁶thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissueculture plates (Corning, Corning N.Y.).

On days 2, 4, and 6 after the fusion, 100 μl of medium is removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,the fusions are screened by ELISA, testing for the presence of mouse IgGthat binds to nGPCR-x. Selected fusion wells are further cloned bydilution until monoclonal cultures producing anti-nGPCR-x antibodies areobtained.

B. Humanization of Anti-nGPCR-x Monoclonal Antibodies

The expression pattern of nGPCR-x as reported herein and the proventrack record of GPCRs as targets for therapeutic intervention suggesttherapeutic indications for nGPCR-x inhibitors (antagonists).nGPCR-x-neutralizing antibodies comprise one class of therapeuticsuseful as nGPCR-x antagonists. Following are protocols to improve theutility of anti-nGPCR-x monoclonal antibodies as therapeutics in humansby “humanizing” the monoclonal antibodies to improve their serumhalf-life and render them less immunogenic in human hosts (i.e., toprevent human antibody response to non-human anti-nGPCR-x antibodies).

The principles of humanization have been described in the literature andare facilitated by the modular arrangement of antibody proteins. Tominimize the possibility of binding complement, a humanized antibody ofthe IgG4 isotype is preferred.

For example, a level of humanization is achieved by generating chimericantibodies comprising the variable domains of non-human antibodyproteins of interest with the constant domains of human antibodymolecules. (See, e.g., Morrison et al., Adv. Immunol., 44:65-92 (1989)).The variable domains of nGPCR-x-neutralizing anti-nGPCR-x antibodies arecloned from the genomic DNA of a B-cell hybridoma or from cDNA generatedfrom mRNA isolated from the hybridoma of interest. The V region genefragments are linked to exons encoding human antibody constant domains,and the resultant construct is expressed in suitable mammalian hostcells (e.g., myeloma or CHO cells).

To achieve an even greater level of humanization, only those portions ofthe variable region gene fragments that encode antigen-bindingcomplementarity determining regions (“CDR”) of the non-human monoclonalantibody genes are cloned into human antibody sequences. (See, e.g.,Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-36 (1988); andTempest et al., Bio/Technology 9: 266-71 (1991)). If necessary, theP-sheet framework of the human antibody surrounding the CDR3 regionsalso is modified to more closely mirror the three dimensional structureof the antigen-binding domain of the original monoclonal antibody. (SeeKettleborough et al., Protein Engin., 4:773-783 (1991); and Foote etal., J. Mol. Biol., 224:487-499 (1992)).

In an alternative approach, the surface of a non-human monoclonalantibody of interest is humanized by altering selected surface residuesof the non-human antibody, e.g., by site-directed mutagenesis, whileretaining all of the interior and contacting residues of the non-humanantibody. See Padlan, Molecular Immunol., 28(4/5):489-98 (1991).

The foregoing approaches are employed using nGPCR-x-neutralizinganti-nGPCR-x monoclonal antibodies and the hybridomas that produce themto generate humanized nGPCR-x-neutralizing antibodies useful astherapeutics to treat or palliate conditions wherein nGPCR-x expressionor ligand-mediated nGPCR-x signaling is detrimental.

C. Human nGPCR-x-Neutralizing Antibodies from Phage Display

Human nGPCR-x-neutralizing antibodies are generated by phage displaytechniques such as those described in Aujame et al., Human Antibodies8(4):155-168 (1997); Hoogenboom, TIBTECH 15:62-70 (1997); and Rader etal., Curr. Opin. Biotechnol. 8:503-508 (1997), all of which areincorporated by reference. For example, antibody variable regions in theform of Fab fragments or linked single chain Fv fragments are fused tothe amino terminus of filamentous phage minor coat protein pIII.Expression of the fusion protein and incorporation thereof into themature phage coat results in phage particles that present an antibody ontheir surface and contain the genetic material encoding the antibody. Aphage library comprising such constructs is expressed in bacteria, andthe library is screened for nGPCR-x-specific phage-antibodies usinglabeled or immobilized nGPCR-x as antigen-probe.

D. Human nGPCR-x-Neutralizing Antibodies from Transgenic Mice

Human nGPCR-x-neutralizing antibodies are generated in transgenic miceessentially as described in Bruggemann et al., Immunol. Today17(8):391-97 (1996) and Bruggemann et al., Curr. Opin. Biotechnol.8:455-58 (1997). Transgenic mice carrying human V-gene segments ingermline configuration and that express these transgenes in theirlymphoid tissue are immunized with a nGPCR-x composition usingconventional immunization protocols. Hybridomas are generated using Bcells from the immunized mice using conventional protocols and screenedto identify hybridomas secreting anti-nGPCR-x human antibodies (e.g., asdescribed above).

Example 10 Assays to Identify Modulators of nGPCR-x Activity

Set forth below are several nonlimiting assays for identifyingmodulators (agonists and antagonists) of nGPCR-x activity. Among themodulators that can be identified by these assays are natural ligandcompounds of the receptor, synthetic analogs and derivatives of naturalligands; antibodies, antibody fragments, and/or antibody-like compoundsderived from natural antibodies or from antibody-like combinatoriallibraries; and/or synthetic compounds identified by high-throughputscreening of libraries; and the like. All modulators that bind nGPCR-xare useful for identifying nGPCR-x in tissue samples (e.g., fordiagnostic purposes, pathological purposes, and the like). Agonist andantagonist modulators are useful for up-regulating and down-regulatingnGPCR-x activity, respectively, to treat disease states characterized byabnormal levels of nGPCR-x activity. The assays may be performed usingsingle putative modulators, and/or may be performed using a knownagonist in combination with candidate antagonists (or visa versa).

A. cAMP Assays

In one type of assay, levels of cyclic adenosine monophosphate (cAMP)are measured in nGPCR-x-transfected cells that have been exposed tocandidate modulator compounds. Protocols for cAMP assays have beendescribed in the literature. (See, e.g., Sutherland et al., Circulation37: 279 (1968); Frandsen et al., Life Sciences 18: 529-541 (1976);Dooley et al., Journal of Pharmacology and Experimental Therapeutics 283(2): 735-41 (1997); and George et al., Journal of Biomolecular Screening2 (4): 23540 (1997)). An exemplary protocol for such an assay, using anAdenylyl Cyclase Activation FlashPlate® Assay from NEN™ Life ScienceProducts, is set forth below.

Briefly, the nGPCR-x coding sequence (e.g., a cDNA or intronless genomicDNA) is subcloned into a commercial expression vector, such as pzeoSV2(Invitrogen), and transiently transfected into Chinese Hamster Ovary(CHO) cells using known methods, such as the transfection protocolprovided by Boehringer-Mannheim when supplying the FuGENE 6 transfectionreagent. Transfected CHO cells are seeded into 96-well microplates fromthe FlashPlate® assay kit, which are coated with solid scintillant towhich antisera to cAMP has been bound. For a control, some wells areseeded with wild type (untransfected) CHO cells. Other wells in theplate receive various amounts of acAMP standard solution for use increating a standard curve.

One or more test compounds (i.e., candidate modulators) are added to thecells in each well, with water and/or compound-free medium/diluentserving as a control or controls. After treatment, cAMP is allowed toaccumulate in the cells for exactly 15 minutes at room temperature. Theassay is terminated by the addition of lysis buffer containing[¹²⁵I]-labeled cAMP, and the plate is counted using a Packard Topcount™96-well microplate scintillation counter. Unlabeled cAMP from the lysedcells (or from standards) and fixed amounts of [¹²⁵I]-cAMP compete forantibody bound to the plate. A standard curve is constructed, and cAMPvalues for the unknowns are obtained by interpolation. Changes inintracellular cAMP levels of cells in response to exposure to a testcompound are indicative of nGPCR-x modulating activity. Modulators thatact as agonists of receptors which couple to the G_(s) subtype of Gproteins will stimulate production of cAMP, leading to a measurable 3-10fold increase in cAMP levels. Agonists of receptors which couple to theG_(i/o) subtype of G proteins will inhibit forskolin-stimulated cAMPproduction, leading to a measurable decrease in cAMP levels of 50-100%.Modulators that act as inverse agonists will reverse these effects atreceptors that are either constitutively active or activated by knownagonists.

B. Aequorin Assays

In another assay, cells (e.g., CHO cells) are transiently co-transfectedwith both a nGPCR-x expression construct and a construct that encodesthe photoprotein apoaquorin. In the presence of the cofactorcoelenterazine, apoaquorin will emit a measurable luminescence that isproportional to the amount of intracellular (cytoplasmic) free calcium.(See generally, Cobbold, et al. “Aequorin measurements of cytoplasmicfree calcium,” In: McCormack J. G. and Cobbold P. H., eds., CellularCalcium: A Practical Approach. Oxford:IRL Press (1991); Stables et al.,Analytical Biochemistry 252: 115-26 (1997); and Haugland, Handbook ofFluorescent Probes and Research Chemicals. Sixth edition. Eugene Oreg.:Molecular Probes (1996).)

In one exemplary assay, nGPCR-x is subcloned into the commercialexpression vector pzeoSV2 (Invitrogen) and transiently co-transfectedalong with a construct that encodes the photoprotein apoaquorin(Molecular Probes, Eugene, Oreg.) into CHO cells using the transfectionreagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocolprovided in the product insert.

The cells are cultured for 24 hours at 37° C. in MEM (Gibco/BRL,Gaithersburg, Md.) supplemented with 10% fetal bovine serum, 2 mMglutamine, 10 U/ml penicillin and 10 μg/ml streptomycin, at which timethe medium is changed to serum-free MEM containing 5 μM coelenterazine(Molecular Probes, Eugene, Oreg.). Culturing is then continued for twoadditional hours at 37° C. Subsequently, cells are detached from theplate using VERSEN (Gibco/BRL), washed, and resuspended at 200,000cells/ml in serum-free MEM.

Dilutions of candidate nGPCR-x modulator compounds are prepared inserum-free MEM and dispensed into wells of an opaque 96-well assay plateat 50 μl/well. Plates are then loaded onto an MLX microtiter plateluminometer (Dynex Technologies, Inc., Chantilly, Va.). The instrumentis programmed to dispense 50 μl cell suspensions into each well, onewell at a time, and immediately read luminescence for 15 seconds. Dosresponse curves for the candidate modulators are constructed using thearea under the curve for each light signal peak. Data are analyzed withSlideWrite, using the equation for a one-site ligand, and EC₅₀ valuesare obtained. Changes in luminescence caused by the compounds areconsidered indicative of modulatory activity. Modulators that act asagonists at receptors which couple to the G_(q) subtype of G proteinsgive an increase in luminescence of up to 100 fold. Modulators that actas inverse agonists will reverse this effect at receptors that areeither constitutively active or activated by known agonists.

C. Luciferase Reporter Gene Assay

The photoprotein luciferase provides another useful tool for assayingfor modulators of nGPCR-x activity. Cells (e.g., CHO cells or COS 7cells) are transiently co-transfected with both a nGPCR-x expressionconstruct (e.g., nGPCR-x in pzeoSV2) and a reporter construct whichincludes a gene for the luciferase protein downstream from atranscription factor binding site, such as the cAMP-response element(CRE), AP-1, or NF-kappa B. Agonist binding to receptors coupled to theG. subtype of G proteins leads to increases in cAMP, thereby activatingthe CRE transcription factor and resulting in expression of theluciferase gene. Agonist binding to receptors coupled to the G_(q)subtype of G protein leads to production of diacylglycerol thatactivates protein kinase C, which activates the AP-1 or NF-kappa Btranscription factors, in turn resulting in expression of the luciferasegene. Expression levels of luciferase reflect the activation status ofthe signaling events. (See generally, George et al, Journal ofBiomolecular Screening 2(4): 235-240 (1997); and Stratowa et al, CurrentOpinion in Biotechnology 6: 574-581 (1995)). Luciferase activity may bequantitatively measured using, e.g., luciferase assay reagents that arecommercially available from Promega (Madison, Wis.).

In one exemplary assay, CHO cells are plated in 24-well culture dishesat a density of 100,000 cells/well one day prior to transfection andcultured at 37° C. in MEM (Gibco/BRL) supplemented with 10% fetal bovineserum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin.Cells are transiently co-transfected with both a nGPCR-x expressionconstruct and a reporter construct containing the luciferase gene. Thereporter plasmids CRE-luciferase, AP-1-luciferase andNF-kappaB-luciferase may be purchased from Stratagene (LaJolla, Calif.).Transfections are performed using the FuGENE 6 transfection reagent(Boehringer-Mannheim) according to the supplier's instructions. Cellstransfected with the reporter construct alone are used as a control.Twenty-four hours after transfection, cells are washed once with PBSpre-warmed to 37° C. Serum-free MEM is then added to the cells eitheralone (control) or with one or more candidate modulators and the cellsare incubated at 37° C. for five hours. Thereafter, cells are washedonce with ice-cold PBS and lysed by the addition of 100 μl of lysisbuffer per well from the luciferase assay kit supplied by Promega. Afterincubation for 15 minutes at room temperature, 15 μl of the lysate ismixed with 50 μl of substrate solution (Promega) in an opaque-white,96-well plate, and the luminescence is read immediately on a Wallacemodel 1450 MicroBeta scintillation and luminescence counter (WallaceInstruments, Gaithersburg, Md.).

Differences in luminescence in the presence versus the absence of acandidate modulator compound are indicative of modulatory activity.Receptors that are either constitutively active or activated by agoniststypically give a 3 to 20-fold stimulation of luminescence compared tocells transfected with the reporter gene alone. Modulators that act asinverse agonists will reverse this effect.

D. Intracellular Calcium Measurement Using FLIPR

Changes in intracellular calcium levels are another recognized indicatorof G protein-coupled receptor activity, and such assays can be employedto screen for modulators of nGPCR-x activity. For example, CHO cellsstably transfected with a nGPCR-x expression vector are plated at adensity of 4×10⁴ cells/well in Packard black-walled, 96-well platesspecially designed to discriminate fluorescence signals emanating fromthe various wells on the plate. The cells are incubated for 60 minutesat 37° C. in modified Dulbecco's PBS (D-PBS) containing 36 mg/L pyruvateand 1 g/L glucose with the addition of 1% fetal bovine serum and one offour calcium indicator dyes (Fluo-3™ AM, Fluo-4™ AM, Calcium Green™-1AM, or Oregon Green™ 488 BAPTA-1 AM), each at a concentration of 4 μM.Plates are washed once with modified D-PBS without 1% fetal bovine serumand incubated for 10 minutes at 37° C. to remove residual dye from thecellular membrane. In addition, a series of washes with modified D-PBSwithout 1% fetal bovine serum is performed immediately prior toactivation of the calcium response.

A calcium response is initiated by the addition of one or more candidatereceptor agonist compounds, calcium ionophore A23187 (10 μM; positivecontrol), or ATP (4 μM; positive control). Fluorescence is measured byMolecular Device's FLIPR with an argon laser (excitation at 488 nm).(See, e.g., Kuntzweiler et al., Drug Development Research, 44(1): 14-20(1998)). The F-stop for the detector camera was set at 2.5 and thelength of exposure was 0.4 milliseconds. Basal fluorescence of cells wasmeasured for 20 seconds prior to addition of candidate agonist, ATP, orA23187, and the basal fluorescence level was subtracted from theresponse signal. The calcium signal is measured for approximately 200seconds, taking readings every two seconds. Calcium ionophore A23187 andATP increase the calcium signal 200% above baseline levels. In general,activated GPCRs increase the calcium signal approximately 10-15% abovebaseline signal.

E. Mitogenesis Assay

In a mitogenesis assay, the ability of candidate modulators to induce orinhibit nGPCR-x-mediated cell division is determined. (See, e.g.,Lajiness et al., Journal of Pharmacology and Experimental Therapeutics267(3): 1573-1581 (1993)). For example, CHO cells stably expressingnGPCR-x are seeded into 96-well plates at a density of 5000 cells/welland grown at 37° C. in MEM with 10% fetal calf serum for 48 hours, atwhich time the cells are rinsed twice with serum-free MEM. Afterrinsing, 80 μl of fresh MEM, or MEM containing a known mitogen, is addedalong with 20 μl MEM containing varying concentrations of one or morecandidate modulators or test compounds diluted in serum-free medium. Ascontrols, some wells on each plate receive serum-free medium alone, andsome receive medium containing 10% fetal bovine serum. Untransfectedcells or cells transfected with vector alone also may serve as controls.

After culture for 16-18 hours, 1 μCi of [³H]-thymidine (2 Ci/mmol) isadded to the wells and cells are incubated for an additional 2 hours at37° C. The cells are trypsinized and collected on filter mats with acell harvester (Tomtec); the filters are then counted in a Betaplatecounter. The incorporation of [³H]-thymidine in serum-free test wells iscompared to the results achieved in cells stimulated with serum(positive control). Use of multiple concentrations of test compoundspermits creation and analysis of dose-response curves using thenon-linear, least squares fit equation: A=B×[C/(D+C)]+G where A is thepercent of serum stimulation; B is the maximal effect minus baseline; Cis the EC₅₀; D is the concentration of the compound; and G is themaximal effect. Parameters B, C and G are determined by Simplexoptimization.

Agonists that bind to the receptor are expected to increase[³H]-thymidine incorporation into cells, showing up to 80% of theresponse to serum. Antagonists that bind to the receptor will inhibitthe stimulation seen with a known agonist by up to 100%.

F. [³⁵S]GTPγS Binding Assay

Because G protein-coupled receptors signal through intracellular Gproteins whose activity involves GTP binding and hydrolysis to yieldbound GDP, measurement of binding of the non-hydrolyzable GTP analog[³⁵S]GTPγS in the presence and absence of candidate modulators providesanother assay for modulator activity. (See, e.g., Kowal et al.,Neuropharmacology 37:179-187 (1998).)

In one exemplary assay, cells stably transfected with a nGPCR-xexpression vector are grown in 10 cm tissue culture dishes tosubconfluence, rinsed once with 5 ml of ice-cold Ca²⁺/Mg²⁺-freephosphate-buffered saline, and scraped into 5 ml of the same buffer.Cells are pelleted by centrifugation (500×g, 5 minutes), resuspended inTEE buffer (25 mM Tris, pH 7.5, 5 in M EDTA, 5 mM EGTA), and frozen inliquid nitrogen. After thawing, the cells are homogenized using a Douncehomogenizer (one ml TEE per plate of cells), and centrifuged at 1,000×gfor 5 minutes to remove nuclei and unbroken cells.

The homogenate supernatant is centrifuged at 20,000×g for 20 minutes toisolate the membrane fraction, and the membrane pellet is washed oncewith TEE and resuspended in binding buffer (20 mM HEPES, pH 7.5, 150 mMNaCl, 10 mM MgCl₂, 1 mM EDTA). The resuspended membranes can be frozenin liquid nitrogen and stored at −70° C. until use.

Aliquots of cell membranes prepared as described above and stored at−70° C. are thawed, homogenized, and diluted into buffer containing 20mM HEPES, 10 mM MgCl, 1 mM EDTA, 120 mM NaCl, 10 μM GDP, and 0.2 mMascorbate, at a concentration of 10-50 μg/ml. In a final volume of 90μl, homogenates are incubated with varying concentrations of candidatemodulator compounds or 100 μM GTP for 30 minutes at 30° C. and thenplaced on ice. To each sample, 10 μl guanosine 5′-O-(3[³⁵S]thio)triphosphate (NEN, 1200 Ci/mmol; [³⁵S]-GTPγS), was added to a finalconcentration of 100-200 pM. Samples are incubated at 30° C. for anadditional 30 minutes, 1 ml of 10 mM HEPES, pH 7.4, 10 mM MgCl₂, at 4°C. is added and the reaction is stopped by filtration.

Samples are filtered over Whatman GF/B filters and the filters arewashed with 20 ml ice-cold 10 mM HEPES, pH 7.4, 10 mM MgCl₂. Filters arecounted by liquid scintillation spectroscopy. Nonspecific binding of[³⁵S]-GTPγS is measured in the presence of 100 μM GTP and subtractedfrom the total. Compounds are selected that modulate the amount of[³⁵]-GTPγS binding in the cells, compared to untransfected controlcells. Activation of receptors by agonists gives up to a five-foldincrease in [³⁵S]GTPγS binding. This response is blocked by antagonists.

G. MAP Kinase Activity Assay

Evaluation of MAP kinase activity in cells expressing a GPCR providesanother assay to identify modulators of GPCR activity. (See, e.g.Lajiness et al., Journal of Pharmacology and Experimental Therapeutics267(3):1573-1581 (1993) and Boulton et al., Cell 65:663-675 (1991).)

In one embodiment, CHO cells stably transfected with nGPCR-x are seededinto 6-well plates at a density of 70,000 cells/well 48 hours prior tothe assay. During this 48-hour period, the cells are cultured at 37° C.in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine,10 U/ml penicillin and 10 μg/ml streptomycin. The cells areserum-starved for 1-2 hours prior to the addition of stimulants.

For the assay, the cells are treated with medium alone or mediumcontaining either a candidate agonist or 200 nM Phorbol ester-myristoylacetate (i.e., PMA, a positive control), and the cells are incubated at37° C. for varying times. To stop the reaction, the plates are placed onice, the medium is aspirated, and the cells are rinsed with 1 ml ofice-cold PBS containing 1 mM EDTA. Thereafter, 200 μl of cell lysisbuffer (12.5 mM MOPS, pH 7.3, 12.5 mM glycerophosphate, 7.5 mM MgCl₂,0.5 MM EGTA, 0.5 mM sodium vanadate, 1 mM benzamidine, 1 mMdithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 μg/mlpepstatin A, and 1 μM okadaic acid) is added to the cells. The cells arescraped from the plates and homogenized by 10 passages through a 23 3/4G needle, and the cytosol fraction is prepared by centrifugation at20,000×g for 15 minutes.

Aliquots (5-10 μl containing 1-5 μg protein) of cytosol are mixed with 1mM MAPK Substrate Peptide (APRTPGGRR (SEQ ID NO: 129), UpstateBiotechnology, Inc., N.Y.) and 50 μM [γ-³²P]ATP (NEN, 3000 Ci/mmol),diluted to a final specific activity of ˜2000 cpm/pmol, in a totalvolume of 25 μl. The samples are incubated for 5 minutes at 30° C., andreactions are stopped by spotting 20 μl on 2 cm² squares of Whatman P81phosphocellulose paper. The filter squares are washed in 4 changes of 1%H₃PO₄, and the squares are subjected to liquid scintillationspectroscopy to quantitate bound label. Equivalent cytosolic extractsare incubated without MAPK substrate peptide, and the bound label fromthese samples are subtracted from the matched samples with the substratepeptide. The cytosolic extract from each well is used as a separatepoint. Protein concentrations are determined by a dye binding proteinassay (Bio-Rad Laboratories). Agonist activation of the receptor isexpected to result in up to a five-fold increase in MAPK enzymeactivity. This increase is blocked by antagonists.

H. [³H]Anachidonic Acid Release

The activation of GPCRs also has been observed to potentiate arachidonicacid release in cells, providing yet another useful assay for modulatorsof GPCR activity. (See, e.g., Kanterman et al., Molecular Pharmacology39:364-369 (1991).) For example, CHO cells that are stably transfectedwith a nGPCR-x expression vector are plated in 24-well plates at adensity of 15,000 cells/well and grown in MEM medium supplemented with10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/mlstreptomycin for 48 hours at 37° C. before use. Cells of each well arelabeled by incubation with [³H]-arachidonic acid (Amersham Corp., 210Ci/mmol) at 0.5 μCi/ml in 1 ml MEM supplemented with 10 mM HEPES, pH7.5, and 0.5% fatty-acid-free bovine serum albumin for 2 hours at 37° C.The cells are then washed twice with 1 ml of the same buffer.

Candidate modulator compounds are added in 1 ml of the same buffer,either alone or with 10 μM ATP and the cells are incubated at 37° C. for30 minutes. Buffer alone and mock-transfected cells are used ascontrols. Samples (0.5 ml) from each well are counted by liquidscintillation spectroscopy. Agonists which activate the receptor willlead to potentiation of the ATP-stimulated release of [³H]-arachidonicacid. This potentiation is blocked by antagonists.

I. Extracellular Acidification Rate

In yet another assay, the effects of candidate modulators of nGFCR-xactivity are assayed by monitoring extracellular changes in pH inducedby the test compounds. (See, e.g., Dunlop et al, Journal ofPharmacological and Toxicological Methods 40(1):47-55 (1998).) In oneembodiment, CHO cells transfected with a nGPCR-x expression vector areseeded into 12 mm capsule cups (Molecular Devices Corp.) at 4×10⁵cells/cup in MEM supplemented with 10% fetal bovine serum, 2 mML-glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin. The cellsare incubated in this medium at 37° C. in 5% CO₂ for 24 hours.

Extracellular acidification rates are measured using a Cytosensormicrophysiometer (Molecular Devices Corp.). The capsule cups are loadedinto the sensor chambers of the microphysiometer and the chambers areperfused with running buffer (bicarbonate-free MEM supplemented with 4mM L-glutamine, 10 units/ml penicillin, 10 μg/ml streptomycin, 26 mMNaCl) at a flow rate of 100 μl/minute. Candidate agonists or otheragents are diluted into the running buffer and perfused through a secondfluid path. During each 60-second pump cycle, the pump is run for 38seconds and is off for the remaining 22 seconds. The pH of the runningbuffer in the sensor chamber is recorded during the cycle from 43-58seconds, and the pump is re-started at 60 seconds to start the nextcycle. The rate of acidification of the running buffer during therecording time is calculated by the Cytosoft program. Changes in therate of acidification are calculated by subtracting the baseline value(the average of 4 rate measurements immediately before addition of amodulator candidate) from the highest rate measurement obtained afteraddition of a modulator candidate. The selected instrument detects 61mV/pH unit. Modulators that act as agonists of the receptor result in anincrease in the rate of extracellular acidification compared to the ratein the absence of agonist. This response is blocked by modulators whichact as antagonists of the receptor.

Example 11 In Situ Hybridization

DNA Probe Preparation for nGPCR-11, -16, 40, -54, and -56

DNA probes for in situ hybridization were prepared as follows. Two setsof primer pairs were prepared. The first set has the sequence for T7polymerase promoter on the 5′ primer to make the sense RNA, and thesecond set has the T7 polymerase promoter sequence on the 3′ primer tomake the antisense RNA. PCR was performed in a 50 μl reaction containing36.5 μl H₂O, 5 μl 10×TT buffer (140 mM Ammonium Sulfate, 0.1% gelatine,0.6 M Tris-tricine pH 8.4), 5 μl 25 mM MgCl₂, 2 μl 10 mM dNTP, 0.4 μlIncyte clone 1722192 DNA, 0.5 μl AmpliTaq (PE Applied Biosystems), and0.3 μl oligo1 (1 mg/ml) and 0.3 μl oligo2 (1 mg/ml) [to make the senseRNA], or 0.3 μl oligo3 (1 mg/ml) and 0.3 μl oligo4 (1 mg/ml) [to makethe antisense RNA]. The PCR reaction involved one cycle at 94° C. for 2min followed by 35 cycles at 94° C. for 30 sec, 60° C. for 30 sec, 72°C. for 30 sec. The two PCR reactions were loaded onto a 1.2% agarosegel. The DNA band was excised from the gel, placed in a GenElute Agarosespin column (Supelco) and spun for 10 min at maximum speed. The elutedDNA was EtOH precipitated and resuspended in transcription buffer. Theprimer sequences for each nGPCR tested are listed below.

For nGPCR-11, the sense primers were:

-   GCGTAATACGACTCACTATAGGGAGACCGCGTGTCTGCTAGACTCTATTTCC 3′(LW1658) (SEQ    ID NO: 159), and:-   5′ TGCCACACTGATGCAACTCC 3′ (LW661) (SEQ ID NO: 160). The antisense    primers were:-   GCGTAATACGACTCACTATAGGGAGACCTGCCACACTGATGCAACTCC (LW1659) SEQ ID    NO: 161) and:-   5′GCGTGTCTGCTAGACTCTATITCC 3′ (LW1660) (SEQ ID NO: 162). The primer    pairs yielded a product of 275 bp.

For nGPCR-16, the sense primers were:

-   5′GCGTAATACGACTCACTATAGGGAGACCGCACGCCACTCTTTACTATCCC (LW1645) (SEQ    ID NO: 163), and:-   5′ GCACAAAACACAATFCCATAAGCC 3′ (LW1648) (SEQ ID NO: 164). The    antisense primers were:-   5′GCGTAATACGACTCACTATAGGGAGACCGCACAAAACACAATTCCATAAGCC 3′ (LW1646)    (SEQ ID NO: 165), and:-   5′ GCTACGCCACTCTTrACTATCCC 3′ (LW1647) (SEQ ID NO: 166). The primer    pairs yielded a product of 283 bp.

For nGPCR-40, the sense primers were:

-   5′GCGTAATACGACTCACTATAGGGAGACCTTATGAGCAGCAATTCATCCC 3′(LW1704) (SEQ    ID NO: 167), and:-   5′CACACCCACCAAGAAATCAG 3′(LW1707) (SEQ ID NO: 168). The antisense    primers were:-   5′GCGTAATACGACTCACTATAGGGAGACCCACACCCACCAAGAAATCAG 3′(LW1705) (SEQ    ID NO: 169), and:-   5′ TTATGAGCAGCAATTCATCCC 3′ (LW1706) (SEQ ID NO: 170). The primer    pairs yielded a product of 25 lbp.

For nGPCR-54, the sense primers were:

-   5′GCGTAATACGACTCACTATAGGGAGACCCGATTATCCACACTTTGACCC 3′ (LW1803) (SEQ    ID NO: 171), and:-   5′CTGAAAGTrGTCGCTGACC 3′ (LW1634) (SEQ ID NO: 172). The anti-sense    primers were:-   GCGTAATACGACTCACTATAGGGAGACCCTGCTGAAAGTTGTCGCTGACC 3′ (LW1804) (SEQ    ID NO: 173), and:-   5′CGATTATCCACACTTTGACGC 3′ (LW1635) (SEQ ID NO: 174). The primer    pairs yielded a product of 286 bp.

For nGPCR-56, the sense primers were:

-   GCGTAATACGACTCACTATAGGGAGACCCTGTAAAATTCACACAAGCACC 3′ (LW1763) (SEQ    ID NO: 175), and:-   5′AGAAGACAGAGCAACCTCC 3′ (L[W1766) (SEQ ID NO: 176). The anti-sense    primers were:-   GCGTAATACGACTCACTATAGGGAGACCAGAAGACAGAGCAACCTCC (LW1764) (SEQ ID    NO: 177) and:-   CTGTAAAATTCACACAAGCACC (LW1765) (SEQ ID NO: 178). The primer pairs    yielded a product of 272 bp.    DNA Probe Preparation For nGPCR-1

Probes for nGPCR-1 were prepared as above with the followingmodifications. Using a sense primer: GCATGGATCCTCTTTTGCTGTATTTCACCCTC)(LW1595) (SEQ ID NO: 179) and an antisense primer:5′GCATGAATTCACAATGCCAGTGATAAGGAAG 3′ (LW1596) (SEQ ID NO: 180), a 271 bpfragment was generated by PCR. The fragment was digested with BamHI andEcoRI and ligated into a BluescriptII vector that had been cut withBamHI and EcoRI. The orientation of the insert was such that T7polymerase generates the anti-sense strand and T3 polymerase generatesthe sense strand.

Histochemistry

Coronal and sagittal oriented rat brain sections were cryosectioned (20μm thick) using a Reichert-Jung cryostat. The individual sections werethaw-mounted onto silanated, nuclease-free slides (CEL Associates, Inc.,Houston, Tex.), and stored at −80° C. The sections were processedstarting with post-fixation in cold 4% paraformaldehyde, rinsed in coldPBS, acetylated using acetic anhydride in triethanolamine buffer anddehydrated through 70%, 95%, and 100% alcohols at room temperature (RT).This was followed with delipidation in chloroform then rehydration in100% and 95% alcohol at room temperature. Sections were air-dried priorto hybridization. Two PCR fragments (˜250 bp) were generated, one thatcontained T7 polymerase on the 5′ end (sense) and the other with T7polymerase on the 3′ end (antisense). The PCR fragments were labeledwith ³⁵S-UTP to yield a specific activity of 0.655×10⁶ cpm/pmol forantisense and 0.675×10⁶ cpm/pmol for sense probe. Both riboprobes weredenatured and added to hybridization buffer containing 50% formamide,10% dextran, 0.3M NaCl, 10 mM Tris, 1 mM EDTA, 1× Denhardts, and 10 mMDTT. Sequential brain cryosections were hybridized with 45 μl/slide ofthe sense and antisense riboprobe hybridization mixture, then coveredwith silanized glass coverslips. The sections were hybridized overnight(15-18 hrs) at 42° C. in an incubator.

Coverslips were washed off the slides in 1×SSC, followed by RNase Atreatment, and high temperature stringency washes (3×, 20 mins at 41°C.) in 0.1×SSC. Slides were dehydrated with 70%, 95% NH₄OAc, and 100%NH₄OAc alcohols, air-dried and exposed to Kodak BioMax MR-1 film. After9 days of exposure, the film was developed. This was followed withcoating selected tissue slides with Kodak NTB-2 nuclear track emulsionand storing the slides in the dark for 23 days. The slides were thendeveloped and counterstained with hematoxylin. Emulsion-coated sectionswere analyzed microscopically to determine the specificity of labeling.Presence of autoradiographic grains (generated by antisense probehybridization) over cell bodies (versus between cell bodies) was used asan index of specific hybridization.

Results

In Situ Hybridization Results Indicated Localization in the FollowingBrain Areas:

nGPCR-1 was localized to the dentate gyrus of hippocampus, piriformcortex, and red nucleus.

nGPCR-11 was localized to the piriform cortex, hippocampus, red nucleus,subthalamic nuclei, dorsal raphe, interpeduncular nucleus, and habenula.nGPCR-16 was localized to the cortex, piriform cortex, hippocampus,thalamus, subthalamic nuclei, hypothalamus, bed nucleus stria terminalsand posterior striatum. nGPCR-40 was localized to the cortex, piriformcortex, hippocampus, substantia nigra compacta, hypothalamus, laterialseptus, bed nucleus stria terminalis, thalamus, ventral tegmental area,interpeduncular nucleus, dorsal raphe, medical geniculate, islands ofCalleja, subthamalmic nuclei, choroid plexus. nGPCR-54 was localized tothe piriform cortex and hippocampus, including the dentate gyms, CA1 andCA3. nGPCR-56 was localized to the piriform cortex, cortex,interpeduncular nuceus, red nucleus, hippocampus, habenula, substantianigra pars compacta, mamillary body stria terminalis, hypothalamus,subthamalmic nuclei, corsal raphe, and ventral tegmental area.

Example 12 Chromosomal Localization

Methods

Chromosomal location of the genes encoding nGPCRs was determined usingthe Stanford G3 Radiation Hybrid Panel (Research Genetics, Inc.,Huntsville, Ala.). This panel contains 83 radiation hybrid clones of theentire human genome created by the Stanford Human Genome Center. PCRreactions were assembled containing 25 ng of DNA from each clone and thecomponents of the Expand Hi-Fi PCR System™ (Roche MolecularBiochemicals, Indianapolis, Ind.) in a final reaction volume of 15 μl.PCR primers were synthesized by Genosys Corp., The Woodlands, Tex. PCRreactions were incubated in a GeneAmp 9700 PCR thermocycler (PerkinElmer Applied Biosystems). The following cycling program was executed:Pre-soak at (940 for 3 min.) (94° for 30 sec.) (52° C. for 60 sec.) (72′for 2 min.)] for 35 cycles. PCR reaction products were then separatedand analyzed by electrophoresis on a 2.0% agarose gel, and stained withethidium bromide. Lanes were scored for the presence or absence of theexpected PCR product and the results submitted to the Stanford HumanGenome Center via e-mail for analysis(http://wwwshgc.stanford.edu./RH/rhserverformnew.html).

nGPCR-40

PCR primers were designed based on the available sequence of the Celerasequence HUM_IDS|Contig|11000258115466. The forward primer used was:

5′ACAGCCCCAAAGCCAAACAC3′ (SEQ ID NO: 181). The reverse primer was:

5′CCGCAGGAGCAATG-AAAATCAG3′ (SEQ ID NO: 182). This prier set will primethe synthesis of a 220 base pair fragment in the presence of theappropriate genomic DNA.

G3 Radiation Hybrid Panel Analysis places nGPCR-40 on chromosome 6, mostnearly linked to Stanford marker SHGC-1836 with a LOD score of 11.84.This marker lies at position 6q21. In a genome scanning data set, Cao etal. (Genomics 1997 Jul. 1: 43(1): 1-8) found excess allele sharing formarkers on 6q13-q26. Greatest allele sharing was at interval 6q21-q22.3with a maximum multipoint MLS value of 3.06 close to marker D6S278.Replication data from a second data set found maximum multipoint MLS atthe interval D6S424-D6S275. These results provide suggestive evidencefor a susceptibility locus for schizophrenia in chromosome 6q from twoindependent data sets.

nGPCR-54

PCR primers were designed based on the available sequence of the Celerasequence GA_(—)11824020. The forward primer used was:

5′CTGTCTCTCTGTCCTCTTCC3′, (SEQ ID NO: 183). The reverse primer used was:

5′GCACCGATCTTCATTGAATTTC3′, (SEQ ID NO: 184). This primer set will primethe synthesis of a 145 base pair fragment in the presence of theappropriate genomic DNA.

G3 Radiation Hybrid Panel Analysis places nGPCR-54 on chromosome 13,most nearly linked to Stanford marker SHGC-68276 with a LOD score of6.31. This marker lies at position 13q32. Numerous investigations havefound significant suggestion of linkage of schizophrenia to this regionof chromosome 13q32. See, for example, Brzustowicz et al., Am J HumGenet 1999 October; 65(4): 1096-1103; Blouinet al., Nat Genet 1998September; 20(1): 70-3; Shaw et al., Am J Med Genet. 1998 Sep. 7; 81(5):364-76; Lin et al., Hum Genet 1997 March, 99(3): 417-20; Pulver et al.,Cold Spring Harb Symp Quant Biol 1996; 61:797-814.

Genes localized to chromosomal regions in linkage with schizophrenia arecandidate genes for disease susceptibility. Genes in these regions withthe potential to play a biochemical/functional role in the diseaseprocess (like G protein coupled receptors) have a high probability ofbeing a disease-modifying locus. nGPCR-40 and -54, because of theirchromosomal location, are attractive targets therefore for screeningligands useful in modulating cellular processes involved inschizophrenia.

Example 13 Clone Deposit Information

In accordance with the Budapest Treaty, clones of the present inventionhave been deposited at the Agricultural Research Culture Collection(NRRL) International Depository Authority, 1815 N. University Street,Peoria, Ill. 61604, U.S.A. Accession numbers and deposit dates areprovided below in Table 6. TABLE 6 DEPOSIT INFORMATION Accession NumberNudapest Treaty Clone NRRL Deposit Date nGPCR-1 (SEQ ID NO:73) B-30243Jan. 18, 2000 nGPCR-5 (SEQ ID NO: 75) B-30244 Jan. 18, 2000 nGPCR-16(SEQ ID NO: 81) B-30245 Jan. 18, 2000 nGPCR-11 (SEQ ID NO: 79) B-30258Feb. 2, 2000 nGPCR-17 (SEQ ID NO: 23) B-30259 Feb. 3, 2000 nGPCR-9 (SEQID NO: 77) B-30262 Feb. 22, 2000 nGPCR-58 (SEQ ID NO: 91) B-30274 Mar.23, 2000 nGPCR-56 (SEQ ID NO: 89) B-30288 May 5, 2000 nGPCR-3 (SEQ IDNO: 185) B-30290 May 5, 2000 nGPCR-54 (SEQ ID NO: 85) B-30291 May 5,2000 nGPCR-40 (SEQ ID NO: 83*) B-30299N Jun. 2, 2000*The clone deposited with NRLL Accession Number N30299N comprises asequence identical to SEQ ID NO: 83 but with the substitution of an “A”at neucleotide position 10.

Example 14 Using nGPCR-x Proteins to Isolate Neurotransmitters

The isolated nGPCR-x proteins can be used to isolate novel or knownneurotransmitters (Saito et al., Nature 400: 265-269, 1999). The cDNAsthat encode the isolated nGPCR-x can be cloned into mammalian expressionvectors and used to stably or transiently transfect mammalian cellsincluding CHO, Cos or HEK293 cells. Receptor expression can bedetermined by Northern blot analysis of transfected cells andidentification of an appropriately sized mRNA band (predicted size fromthe cDNA). Brain regions shown by mRNA analysis to express each of thenGPCR-x proteins could be processed for peptide extraction using any ofseveral protocols ((Reinsheidk R. K. et al., Science 270: 243-247, 1996;Sakurai, T., et al., Cell 92; 573-585, 1998; Hinuma, S., et al., Nature398: 272-276, 1998). Chromotographic fractions of brain extracts couldbe tested for ability to activate nGPCR-x proteins by measuring secondmessenger production such as changes in cAMP production in the presenceor absence of forskolin, changes in inositol 3-phosphate levels, changesin intracellular calcium levels or by indirect measures of receptoractivation including receptor stimulated mitogenesis, receptor mediatedchanges in extracellular acidification or receptor mediated changes inreporter gene activation in response to cAMP or calcium (these methodsshould all be referenced in other sections of the patent). Receptoractivation could also be monitored by co-transfecting cells with achimeric GI_(q/i3) to force receptor coupling to a calcium stimulatingpathway (Conklin et al., Nature 363; 274-276, 1993). Neurotransmittermediated activation of receptors could also be monitored by measuringchanges in [³⁵S]-GTPKS binding in membrane fractions prepared fromtransfected mammalian cells. This assay could also be performed usingbaculoviruses containing nGPCR-x proteins infected into SF9 insectcells.

The neurotransmitter which activates nGPCR-x proteins can be purified tohomogeneity through successive rounds of purification using nGPCR-xproteins activation as a measurement of neurotransmitter activity. Thecomposition of the neurotransmitter can be determined by massspectrometry and Edman degradation if peptidergic. Neurotramittersisolated in this manner will be bioactive materials which will alterneurotransmission in the central nervous system and will producebehavioral and biochemical changes.

Example 15 Using nGPCR-x Proteins to Isolate and Purify G Proteins

cDNAs encoding nGPCR-x proteins are epitope-tagged at the amino terminusend of the cDNA with the cleavable influenza-hemagglutinin signalsequence followed by the FLAG epitope (IBI, New Haven, Conn.).Additionally, these sequences are tagged at the carboxyl terminus withDNA encoding six histidine residues. (Amino and Carboxyl TerminalModifications to Facilitate the Production and Purification of a GProtein-Coupled Receptor, B. K. Kobilka, Analytical Biochemistry, Vol.231, No. 1, October 1995, pp. 269-271). The resulting sequences arecloned into a baculovirus expression vector such as pVL1392(Invitrogen). The baculovirus expression vectors are used to infect SF-9insect cells as described (Guan, X. M., Kobilka, T. S., and Kobilka, B.K. (1992) J. Biol. Chem. 267, 21995-21998). Infected SF-9 cells could begrown in 1000-ml cultures in SF900 II medium (Life Technologies, Inc.)containing 5% fetal calf serum (Gemini, Calabasas, Calif.) and 0.1 mg/mlgentamicin (Life Technologies, Inc.) for 48 hours at which time thecells could be harvested. Cell membrane preparations could be separatedfrom soluble proteins following cell lysis. nGPCR-x protein purificationis carried out as described for purification of the θ2 receptor(Kobilka, Anal. Biochem., 231 (1): 269-271, 1995) includingsolubilization of the membranes in 0.8-1.0% n-dodecyl-D-maltoside (DM)(CalBiochem, La Jolla, Calif.) in buffer containing protease inhibitorsfollowed by Ni-column chromatography using chelating Sepharose™(Pharmacia, Uppsala, Sweden). The eluate from the Ni-column is furtherpurified on an M1 anti-FLAG antibody column (IBI). Receptor containingfractions are monitored by using receptor specific antibodies followingwestern blot analysis or by SDS-PAGE analysis to look for an appropriatesized protein band (appropriate size would be the predicted molecularweight of the protein).

This method of purifying G protein is particularly useful to isolate Gproteins that bind to the nGPCR-x proteins in the absence of anactivating ligand.

Some of the preferred embodiments of the invention described above areoutlined below and include, but are not limited to, the followingembodiments. As those skilled in the art will appreciate, numerouschanges and modifications may be made to the preferred embodiments ofthe invention without departing from the spirit of the invention. It isintended that all such variations fall within the scope of theinvention.

The entire disclosure of each publication cited herein is herebyincorporated by reference.

1. An isolated nucleic acid molecule comprising a nucleotide sequencethat encodes a polypeptide comprising an amino acid sequence homologousto a sequence of SEQ ID NO: 192, and fragments thereof; said nucleicacid molecule encoding at least a portion of nGPCR-14, wherein theisolated nucleic acid comprises at least one or more nucleotides fromone or more of the following regions of SEQ ID NO: 191: nucleotides 1 to193 of SEQ ID NO: 191; nucleotides 612 to 644 of SEQ ID NO:191;nucleotides 697 to 706 of SEQ ID NO:191; nucleotides 1011 to 1049 of SEQID NO: 191; nucleotides 1051 to 1057 of SEQ ID NO:191; nucleotides 1090to 1096 of SEQ ID NO:191; or nucleotides 1141 to 1642 of SEQ ID NO:191.2. The isolated nucleic acid molecule of claim 1 comprising a sequencethat encodes a polypeptide comprising a sequences of SEQ ID NO: 192, andfragments thereof.
 3. The isolated nucleic acid molecule of claim 1comprising a sequence homologous to a sequence of SEQ ID NO: 191, andfragments thereof.
 4. The isolated nucleic acid molecule of claim 1comprising a sequence of SEQ ID NO:191, and fragments thereof.
 5. Theisolated nucleic acid molecule of claim 1 wherein said nucleic acidmolecule is DNA.
 6. The isolated nucleic acid molecule of claim 1wherein said nucleic acid molecule is RNA.
 7. An expression vectorcomprising a nucleic acid molecule of any one of claims 1 to
 5. 8. Theexpression vector of claim 7 wherein said nucleic acid moleculecomprises a sequence of SEQ ID NO:191.
 9. The expression vector of claim7 wherein said vector is a plasmid.
 10. The expression vector of claim 7wherein said vector is a viral particle.
 11. The expression vector ofclaim 10 wherein said vector is selected from the group consisting ofadenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses,adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, andretroviruses.
 12. The expression vector of claim 7 wherein said nucleicacid molecule is operably connected to a promoter selected from thegroup consisting of simian virus 40, mouse mammary tumor virus, longterminal repeat of human immunodeficiency virus, maloney virus,cytomegalovirus immediate early promoter, Epstein Barr virus, roussarcoma virus, human actin, human myosin, human hemoglobin, human musclecreatine, and human metalothionein.
 13. A host cell transformed with anexpression vector of claim
 7. 14. The transformed host cell of claim 13wherein said cell is a bacterial cell.
 15. The transformed host cell ofclaim 14 wherein said bacterial cell is E. coli.
 16. The transformedhost cell of claim 13 wherein said cell is yeast.
 17. The transformedhost cell of claim 16 wherein said yeast is S. cerevisiae.
 18. Thetransformed host cell of claim 13 wherein said cell is an insect cell.19. The transformed host cell of claim 18 wherein said insect cell is S.frugiperda.
 20. The transformed host cell of claim 13 wherein said cellis a mammalian cell.
 21. The transformed host cell of claim 20 whereinmammalian cell is selected from the group consisting of chinese hamsterovary cells, HeLa cells, African green monkey kidney cells, human 293cells, and murine 3T3 fibroblasts.
 22. An isolated nucleic acid moleculecomprising a nucleotide sequence complementary to at least a portion ofa sequence of SEQ ID NO:191, said portion comprising at least 10nucleotides, wherein said isolated nucleic acid comprises at least oneor more nucleotides from one or more of the following regions of SEQ IDNO: 191: nucleotides 1 to 193 of SEQ ID NO: 191; nucleotides 612 to 644of SEQ ID NO:191; nucleotides 697 to 706 of SEQ ID NO: 191; nucleotides1011 to 1049 of SEQ ID NO: 191; nucleotides 1051 to 1057 of SEQ IDNO:191; nucleotides 1090 to 1096 of SEQ ID NO:191; or nucleotides 1141to 1642 of SEQ ID NO:191.
 23. The nucleic acid molecule of claim 22wherein said molecule is an antisense oligonucleotide directed to aregion of a sequence of SEQ ID NO:
 191. 24. The nucleic acid molecule ofclaim 23 wherein said oligonucleotide is directed to a regulatory regionof a sequence of SEQ ID NO:
 191. 25. A composition comprising a nucleicacid molecule of any one of claims 1 to 5 or 22 and an acceptablecarrier or diluent.
 26. A composition comprising a recombinantexpression vector of claim 7 and an acceptable carrier or diluent.
 27. Amethod of producing a polypeptide that comprises a sequence of SEQ IDNO: 192, and homologs and fragments thereof, said method comprising thesteps of: a) introducing a recombinant expression vector of claim 7 intoa compatible host cell; b) growing said host cell under conditions forexpression of said polypeptide; and c) recovering said polypeptide,wherein the polypeptide comprising a sequence of SEQ ID NO:192 comprisesat least one or more amino acid residues from one or more of thefollowing regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQID NO:192; amino acid residues 68 to 77 of SEQ ID NO:192; amino acidresidues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513of SEQ ID NO:
 192. 28. The method of claim 27 wherein said host cell islysed and said polypeptide is recovered from the lysate of said hostcell.
 29. The method of claim 27 wherein said polypeptide is recoveredby purifying the culture medium without lysing said host cell.
 30. Anisolated polypeptide encoded by a nucleic acid molecule of claim 1,wherein the polynucleotide comprises at least one or more amino acidresidues from one or more of the following regions of SEQ ID NO: 192:amino acid residues 1 to 42 of SEQ ID NO:192; amino acid residues 68 to77 of SEQ ID NO:192; amino acid residues 185 to 197 of SEQ ID NO: 192;or amino acid residues 293 to 513 of SEQ ID NO:
 192. 31. The polypeptideof claim 30 wherein said polypeptide comprises a fragment of SEQ IDNO:192.
 32. The polypeptide of claim 30 wherein said polypeptidecomprises an amino acid sequence homologous to a sequence of SEQ ID NO:192.
 33. The polypeptide of claim 30 wherein said sequence homologous toa sequence of SEQ ID NO:192 comprises at least one conservative aminoacid substitution compared to the sequence of SEQ ID NO:
 192. 34. Thepolypeptide of claim 30 wherein said polypeptide comprises a fragment ofa polypeptide with a sequence of SEQ ID NO:192.
 35. A compositioncomprising a polypeptide of claim 30 and an acceptable carrier ordiluent.
 36. An isolated antibody which binds to an epitope on apolypeptide of claim
 30. 37. The antibody of claim 36 wherein saidantibody is a monoclonal antibody.
 38. A composition comprising anantibody of claim 36 and an acceptable carrier or diluent.
 39. A methodof inducing an immune response in a mammal against a polypeptide ofclaim 30 comprising administering to said mammal an amount of saidpolypeptide sufficient to induce said immune response.
 40. A method foridentifying a compound which binds nGPCR-14 comprising the steps of: a)contacting nGPCR-14 with a compound; and b) determining whether saidcompound binds nGPCR-14.
 41. The method of claim 40 wherein the nGPCR-14comprises an amino acid sequence of SEQ ID NO:
 192. 42. The method ofclaim 40 wherein binding of said compound to nGPCR-14 is determined by aprotein binding assay.
 43. The method of claim 40 wherein said proteinbinding assay is selected from the group consisting of a gel-shiftassay, Western blot, radiolabeled competition assay, phage-basedexpression cloning, co-fractionation by chromatography,co-precipitation, cross linking, interaction trap/two-hybrid analysis,southwestern analysis, and ELISA.
 44. A compound identified by themethod of claim
 40. 45. A method for identifying a compound which bindsa nucleic acid molecule encoding nGPCR-14 comprising the steps of: a)contacting said nucleic acid molecule encoding nGPCR-14 with a compound;and b) determining whether said compound binds said nucleic acidmolecule.
 46. The method of claim 45 wherein binding is determined by agel-shift assay.
 47. A compound identified by the method of claim 45.48. A method for identifying a compound which modulates the activity ofnGPCR-14 comprising the steps of: a) contacting nGPCR-14 with acompound; and b) determining whether nGPCR-14 activity has beenmodulated.
 49. The method of claim 48 wherein the nGPCR-14 comprises anamino acid sequence of SEQ ID NO:
 192. 50. The method of claim 48wherein said activity is neuropeptide binding.
 51. The method of claim48 wherein said activity is neuropeptide signaling.
 52. A compoundidentified by the method of claim
 48. 53. A method of identifying ananimal homolog of nGPCR-14 comprising the steps: a) comparing thenucleic acid sequences of the animal with a sequence of SEQ ID NO: 191,and portions thereof, said portions being at least 10 nucleotides; andb) identifying nucleic acid sequences of the animal that are homologousto said sequence of SEQ ID NO: 191, and portions thereof.
 54. The methodof claim 53 wherein comparing the nucleic acid sequences of the animalwith a sequence selected of SEQ ID NO: 191, and portions thereof, saidportions being at least 10 nucleotides, is performed by DNAhybridization.
 55. The method of claim 53 wherein comparing the nucleicacid sequences of the animal with a sequence selected of SEQ ID NO: 191,and portions thereof, said portions being at least 10 nucleotides, isperformed by computer homology search.
 56. A method of screening a humansubject to diagnose a disorder affecting the brain or geneticpredisposition therefor, comprising the steps of: (a) assaying nucleicacid of a human subject to determine a presence or an absence of amutation altering an amino acid sequence, expression, or biologicalactivity of at least one nGPCR that is expressed in the brain, whereinthe nGPCR comprises an amino acid sequence of SEQ ID NO:192, and allelicvariants thereof, and wherein the nucleic acid corresponds to a geneencoding the nGPCR; and (b) diagnosing the disorder or predispositionfrom the presence or absence of said mutation, wherein the presence of amutation altering the amino acid sequence, expression, or biologicalactivity of the nGPCR in the nucleic acid correlates with an increasedrisk of developing the disorder.
 57. A method according to claim 56,wherein the nGPCR is nGPCR-14 comprising an amino acid sequence setforth in SEQ ID NO:192 or an allelic variant thereof.
 58. A methodaccording to claim 56, wherein the assaying step comprises at least oneprocedure selected from the group consisting of: a) comparing nucleotidesequences from the human subject and reference sequences and determininga difference of at least a nucleotide of at least one codon between isthe nucleotide sequences from the human subject that encodes an nGPCR-14allele and an nGPCR-14 reference sequence; (b) performing ahybridization assay to determine whether nucleic acid from the humansubject has a nucleotide sequence identical to or different from one ormore reference sequences; (c) performing a polynucleotide migrationassay to determine whether nucleic acid from the human subject has anucleotide sequence identical to or different from one or more referencesequences; and (d) performing a restriction endonuclease digestion todetermine whether nucleic acid from the human subject has a nucleotidesequence identical to or different from one or more reference sequences.59. A method according to claim 58 wherein the assaying step comprises:performing a polymerase chain reaction assay to amplify nucleic acidcomprising nGPCR-14 coding sequence, and determining nucleotide sequenceof the amplified nucleic acid.
 60. A method of screening for an nGPCR-14mental disorder genotype in a human patient, comprising the steps of:(a) providing a biological sample comprising nucleic acid from saidpatient, said nucleic acid including sequences corresponding to allellesof nGPCR-14; and (b) detecting the presence of one or more mutations inthe nGPCR-14 allelle; wherein the presence of a mutation in an nGPCR-40allelle or nGPCR-54 allele is indicative of a mental disorder genotype.61. The method according to claim 59 wherein said biological sample is acell sample.
 62. The method according to claim 59 wherein said detectingthe presence of a mutation comprises sequencing at least a portion ofsaid nucleic acid, said portion comprising at least one codon of saidnGPCR-14 alleles.
 63. The method according to claim 59 wherein saidnucleic acid is DNA.
 64. The method according to claim 59 wherein saidnucleic acid is RNA.
 65. A kit for screening a human subject to diagnosea mental disorder or a genetic predisposition therefor, comprising, inassociation: (a) an oligonucleotide useful as a probe for identifyingpolymorphisms in a human nGPCR-14 gene, the oligonucleotide comprising6-50 nucleotides in a sequence that is identical or complementary to asequence of a wild type human nGPCR-14 gene sequence or nGPCR-14 codingsequence, except for one sequence difference selected from the groupconsisting of a nucleotide addition, a nucleotide deletion, ornucleotide substitution; and (b) a media packaged with theoligonucleotide, said media containing information for identifyingpolymorphisms that correlate with schizophrenia or a geneticpredisposition therefor, the polymorphisms being identifiable using theoligonucleotide as a probe.
 66. A method of identifying a nGPCR allelicvariant that correlates with a mental disorder, comprising steps of: (a)providing a biological sample comprising nucleic acid from a humanpatient diagnosed with a mental disorder, or from the patient's geneticprogenitors or progeny; (b) detecting in the nucleic acid the presenceof one or more mutations in an nGPCR that is expressed in the brain,wherein the nGPCR comprises an amino acid sequence of SEQ ID NO: 192,and allelic variants thereof, and wherein the nucleic acid includessequence corresponding to the gene or genes encoding nGPCR; wherein theone or more mutations detected indicates an allelic variant thatcorrelates with a mental disorder.
 67. A method according to claim 66wherein the at least one nGPCR is nGPCR-14, or an allelic variantthereof.
 68. A purified and isolated polynucleotide comprising anucleotide sequence encoding a nGPCR-14 allelic variant identifiedaccording to claim
 67. 69. A host cell transformed or transfected with apolynucleotide according to claim 68 or with a vector comprising thepolynucleotide.
 70. A purified polynucleotide comprising a nucleotidesequence encoding nGPCR-14 of a human with a mental disorder; whereinsaid polynucleotide hybridizes to the complement of SEQ ID NO:191 underthe following hybridization conditions: (a) hybridization for 16 hoursat 42° C. in a hybridization solution comprising 50% formamide, 1% SDS,1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at60° C. in a wash solution comprising 0.1×SSC and 1% SDS; and wherein thepolynucleotide that encodes the nGPCR-14 amino acid sequence of thehuman differs from SEQ ID NO:192 by at least one residue and comprisesat least one or more amino acid residues from one or more of thefollowing regions of SEQ ID NO: 192: amino acid residues 1 to 42 of SEQID NO: 192; amino acid residues 68 to 77 of SEQ ID NO: 192; amino acidresidues 185 to 197 of SEQ ID NO: 192; or amino acid residues 293 to 513of SEQ ID NO:
 192. 71. A vector comprising a polynucleotide according toclaim
 70. 72. A host cell that has been transformed or transected with apolynucleotide according to claim 70 and that expresses the nGPCR-14protein encoded by the polynucleotide.
 73. A host cell according toclaim 72 that has been co-transfected with a polynucleotide encoding thenGPCR-14 amino acid sequence set forth in SEQ ID NO:192 and thatexpresses the nGPCR-14 protein having the amino acid sequence set forthin SEQ ID NO:192.
 74. A method for identifying a modulator of biologicalactivity of nGPCR-14 comprising the steps of: a) contacting a cellaccording to claim 72 in the presence and in the absence of a putativemodulator compound; b) measuring nGPCR-14 biological activity in thecell; wherein decreased or increased nGPCR-14 biological activity in thepresence versus absence of the putative modulator is indicative of amodulator of biological activity.
 75. A method to identify compoundsuseful for the treatment of a mental disorder, said method comprisingsteps of: (a) contacting a composition comprising nGPCR-14 with acompound suspected of binding nGPCR-14; (b) detecting binding betweennGPCR-14 and the compound suspected of binding nGPCR-14; whereincompounds identified as binding nGPCR-14 are candidate compounds usefulfor the treatment of a mental disorder.
 76. A method for identifying acompound useful as a modulator of binding between nGPCR-14 and a bindingpartner of nGPCR-14 comprising the steps of: (a) contacting the bindingpartner and a composition comprising nGPCR-14 in the presence and in theabsence of a putative modulator compound; (b) detecting binding betweenthe binding partner and nGPCR-14; wherein decreased or increased bindingbetween the binding partner and nGPCR-14 in the presence of the putativemodulator, as compared to binding in the absence of the putativemodulator is indicative a modulator compound useful for the treatment ofschizophrenia.
 77. A method according to claim 75 or 76 wherein thecomposition comprises a cell expressing nGPCR-14 on its surface.
 78. Amethod according to claim 77 wherein the composition comprises a celltransformed or transfected with a polynucleotide that encodes nGPCR-14.79. A method of purifying a G protein from a sample containing said Gprotein comprising the steps of: a) contacting said sample with apolypeptide of claim 1 for a time sufficient to allow said G protein toform a complex with said polypeptide; b) isolating said complex fromremaining components of said sample; c) maintaining said complex underconditions which result in dissociation of said G protein from saidpolypeptide; and d) isolating said G protein from said polypeptide. 80.The method of claim 79 wherein said sample comprises an amino acidsequence of SEQ ID NO:
 192. 81. The method of claim 79 wherein saidpolypeptide comprises an amino acid sequence homologous to a sequence ofSEQ ID NO:192.